Acetyl-coa carboxylase variants

ABSTRACT

The disclosure relates to acetyl-CoA carboxylase (ACC) variants and host cells expressing them for the production of malonyl-CoA derived compounds including fatty acid derivatives. Further contemplated are methods of producing increased amounts of malonyl-CoA derived compounds and related cell cultures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/021,515, filed Mar. 11, 2016, which is a U.S. National Stage under 35U.S.C. § 371 of International Application Serial No. PCT/US2014/055510,filed Sep. 12, 2014, which claims the benefit of U.S. ProvisionalApplication No. 61/877,418, filed Sep. 13, 2013, and U.S. ProvisionalApplication No. 61/892,242, filed Oct. 17, 2013, the entire disclosuresof which are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 12, 2014, isnamed LS00050PCT_SL.txt and is 128,259 bytes in size.

FIELD

The disclosure relates to acetyl-CoA carboxylase (ACC) variants forproduction of a malonyl-CoA derived compound including a fatty acidderivative. Further contemplated are host cells that express the ACCvariants and related cell cultures. Still encompassed are methods ofproducing malonyl-CoA derived compounds by employing the host cellsexpressing the ACC variants.

BACKGROUND

Petroleum is a limited, natural resource found in the earth in liquid,gaseous, or solid forms. However, petroleum products are developed atconsiderable costs, both financial and environmental. In its naturalform, crude petroleum extracted from the Earth has few commercial uses.It is a mixture of hydrocarbons, e.g., paraffins (or alkanes), olefins(or alkenes), alkynes, napthenes (or cylcoalkanes), aliphatic compounds,aromatic compounds, etc. of varying length and complexity. In addition,crude petroleum contains other organic compounds (e.g., organiccompounds containing nitrogen, oxygen, sulfur, etc.) and impurities(e.g., sulfur, salt, acid, metals, etc.). Due to its high energy densityand its easy transportability, most petroleum is refined into fuels,such as transportation fuels (e.g., gasoline, diesel, aviation fuel,etc.), heating oil, liquefied petroleum gas, etc.

Petrochemicals can be used to make specialty chemicals, such asplastics, resins, fibers, elastomers, pharmaceuticals, lubricants, orgels. Specialty chemicals have many commercial uses. Examples ofspecialty chemicals which can be produced from petrochemical rawmaterials include fatty acids, hydrocarbons (e.g., long chainhydrocarbons, branched chain hydrocarbons, saturated hydrocarbons,unsaturated hydrocarbons, etc.), fatty alcohols, fatty esters, fattyaldehydes, ketones, lubricants, etc. Fatty acids are used commerciallyas surfactants. Surfactants can be found, for example, in detergents andsoaps. Fatty acids can also be used as additives in fuels, lubricatingoils, paints, lacquers, candles, shortenings, cosmetics, andemulsifiers. In addition, fatty acids are used as accelerator activatorsin rubber products. Fatty acids can also be used as a feedstock toproduce methyl esters, amides, amines, acid chlorides, anhydrides,ketene dimers, peroxy acids and esters.

Fatty esters have many commercial uses. For example, biodiesel, analternative fuel, is comprised of esters (e.g., fatty acid methyl ester(FAME), fatty acid ethyl esters (FAEE), etc.). Some low molecular weightesters are volatile with a pleasant odor which makes them useful asfragrances or flavoring agents. In addition, esters are used as solventsfor lacquers, paints, and varnishes. Furthermore, some naturallyoccurring substances, such as waxes, fats, and oils are comprised ofesters. Esters are also used as softening agents in resins and plastics,plasticizers, flame retardants, and additives in gasoline and oil. Inaddition, esters can be used in the manufacture of polymers, films,textiles, dyes, and pharmaceuticals.

Similarly, fatty alcohols have numerous commercial uses. For example,worldwide annual sales of fatty alcohols and their derivatives are inexcess of US$1 billion. The shorter chain fatty alcohols are used in thecosmetic and food industries as emulsifiers, emollients, and thickeners.Due to their amphiphilic nature, fatty alcohols behave as nonionicsurfactants, which are useful in personal care and household products,for example, detergents. In addition, fatty alcohols are used in waxes,gums, resins, pharmaceutical lotions, lubricating oil additives, textileantistatic and finishing agents, plasticizers, cosmetics, industrialsolvents, and solvents for fats.

Acetyl CoA carboxylase (ACC) plays an important role in regulating fattyacid synthesis and degradation. It is a biotin-dependent enzyme complexthat catalyzes the first committed step of fatty acid biosynthesis,i.e., the irreversible carboxylation of acetyl-CoA to malonyl-CoA. ACCproduces malonyl-CoA via its two catalytic activities, i.e., biotincarboxylase (BC) and carboxyltransferase (CT). In most prokaryotes, ACCis a multi-subunit enzyme that includes four polypeptides (subunits),encoded by distinct genes whose coordinate expression is controlledthrough multiple levels of regulation (Cronan et al. (2002) Progress inLipid Research 41:407-435; James et al. (2004) Journal of BiologicalChemistry 279(4):2520-2527). The four polypeptides of ACC assemble intoa complex at a fixed ratio (Broussard et al. (2013) Structure21:650-657). More specifically, the ACC reaction requires four proteins,i.e., biotin carboxylase (BC), biotinoyl (or biotin) carboxyl carrierprotein (BCCP), and two proteins that form the carboxyltransferase (CT).The overall ACC reaction can be assayed by the ATP-dependent conversionof the acid-labile NaH¹⁴CO₃ to the acid-stable malonic acid. There aresimilarities and differences between the ACC subunits of bacteria andplant plastids. But despite the complexity of the plant proteins, thesequences that are essential for ACC activity are not significantlydifferent from the bacterial homologues (Cronan et al., supra).

It has been reported that the E. coli ACC is the least stable of theknown ACC enzymes. The overall activity can be measured only when allfour subunits are present at high concentrations, although two partialreactions can be measured in dilute protein solutions. The stablecomplexes are believed to be the BC complex and the CT alpha₂ beta₂complex. The full length BCCP has been purified as a dimer and there arehints of the presence of an unstable BC₂-BCCP₂ complex. Other bacterialACCs seem more stable than that of E. coli and ACC activity can bemeasured in dilute extracts of Helicobacter pylori and Pseudomonascitronellolis. In addition, the plant plastid ACCs seem more stable thanE. coli ACCs. However, as in E. coli further purification of the intactenzyme results in dissociation and loss of the ACC activity that can berestored by mixing fractions containing the partial reaction activities.The subcomplexes are BC-BCCP and CT with no evidence for free intactBCCP or free CT beta, suggesting that BCCP and CT beta are degraded whenfree in solution (Cronan et al., supra).

The identification of the E. coli acc genes including accA, accB, accC,and accD has facilitated the study of the ACC proteins. Radiationsuicide selections have been used to isolate mutants in fatty acidsynthesis including in genes accB and accD that encode ACC subunits BCCPand CT beta, respectively. The accB mutant has been studied moreextensively and the mutation G133S is responsible for temperaturesensitive growth. This mutation results in a steric clash within thebiotinoyl domain. This resultant mutant protein is easily denatured athigher temperatures and is thus sensitive to intracellular proteases.The mutant BCCP strain has only about 25 percent of the normal level ofBCCP when it is grown at 30° C., yet the rates of growth and fatty acidsynthesis are normal (Cronan et al., supra). It is, however, known thatincreasing the concentration of all four proteins of ACC can improve theflux through fatty acid biosynthesis to a certain degree (Davis et al.(2000) Journal of Biological Chemistry 275(37):28593-28598). Conversely,it has been shown that E. coli ACC can be inhibited by acylatedderivatives of ACP while ACP lacking an acyl moiety cannot inhibit ACC(Davies et al. (2001) Journal of Bacteriology 183 (4): 1499-1503).

There is a need for alternative routes to create both fuels and productscurrently derived from petroleum. As such, microbial systems offer thepotential for the biological production of numerous types of biofuelsand chemicals. Renewable fuels and chemicals can be derived fromgenetically engineered organisms (such as bacteria, yeast and algae).Naturally occurring biosynthetic pathways can be genetically altered toenable engineered organisms to synthesize renewable fuel and chemicalproducts. In addition, microbes can be tailored, or metabolicallyengineered, to utilize various carbon sources as feedstock for theproduction of fuel and chemical products. Thus, it would be desirable toengineer an ACC to produce higher yields of malonyl-derived compounds(e.g., fatty esters, fatty alcohols and other fatty acid derivatives aswell as non-fatty acid compounds) when expressed in a recombinant hostcell.

Notwithstanding the advances in the field, there remains a need forimprovements in genetically modified enzymes, recombinant host cells,methods and systems in order to achieve robust and cost-effectiveproduction of fuels and chemicals through fermentation of recombinanthost cells. The present disclosure addresses this need by providing ACCvariants that increase the yield and titer of malonyl-derived compounds.

SUMMARY

One aspect of the disclosure provides a variant biotin carboxyl carrierprotein (BCCP) having at least one mutation in its amino acid sequence.In one particular aspect the disclosure provides a variant biotincarboxyl carrier protein (BCCP) comprising at least one mutation in itsamino acid sequence, wherein the variant BCCP has a polypeptide sequencefrom any one or more of the following sequence identifying numbersincluding SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90. In oneembodiment, the variant BCCP confers to a cell an increased productionof a malonyl-CoA-derived compound when compared to a corresponding wildtype cell. In another embodiment, the variant BCCP may confer improvedacetyl-CoA carboxylase (ACC) activity when expressed in a cell,resulting in increased production of a malonyl-CoA-derived compound whencompared to a corresponding wild type cell. The malonyl-CoA-derivedcompound includes, but is not limited to, a fatty acid derivative suchas a free fatty acid, a fatty acid methyl ester (FAME), a fatty acidethyl ester (FAEE), a fatty alcohol, a fatty amine, a beta hydroxy fattyacid derivative, a bifunctional fatty acid derivative (e.g., w-hydroxyfatty acid, w-hydroxy diol, w-hydroxy FAME, w-hydroxy FAEE), anunsaturated fatty acid derivative, as well as a non-fatty acid basedcompound such as a flavanone and/or a flavonoid, a polyketide, and3-hydroxypropionic acid.

Another aspect of the disclosure provides a variant biotin carboxylcarrier protein (BCCP) having at least one mutation in its amino acidsequence, wherein the mutation is in the N-terminal amino acid region.In one embodiment, the mutation is in amino acid position 2 of SEQ IDNO: 2. In another embodiment, the variant BCCP confers to a cell anincreased production of a malonyl-CoA-derived compound when compared toa corresponding wild type cell. In another embodiment, the variant BCCPmay confer improved acetyl-CoA carboxylase (ACC) activity when expressedin a cell, resulting in increased production of a malonyl-CoA-derivedcompound when compared to a corresponding wild type cell.

Another aspect of the disclosure provides a variant biotin carboxylcarrier protein (BCCP) having at least one mutation in its amino acidsequence, wherein the variant BCCP is selected from SEQ ID NOS: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88 and/or 90. In one embodiment, the variant BCCP confers toa cell an increased production of a malonyl-CoA-derived compound whencompared to a corresponding wild type cell. In another embodiment, thevariant BCCP may confer improved acetyl-CoA carboxylase (ACC) activitywhen expressed in a cell, resulting in increased production of amalonyl-CoA-derived compound when compared to a corresponding wild typecell.

Still another aspect of the disclosure provides a variant BCCP that isencoded by a variant accB gene or accB nucleic acid sequence, whereinthe nucleic acid sequence is selected from SEQ ID NOS: 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87 and/or 89.

Another aspect of the disclosure provides a recombinant cell orrecombinant microorganism that expresses a variant BCCP, wherein thevariant BCCP has at least one mutation in its amino acid sequence. Inone embodiment, the cell is a host cell. In another embodiment, the cellis a microbial cell or microbial host cell. In another embodiment, themicroorganism is a microbial cell or microbial host cell or a microbe.In one embodiment, the mutation is in the N-terminal amino acid region.In another embodiment, the mutation is in amino acid position 2 of SEQID NO: 2. In another embodiment, the mutation is a substitution. Invarious embodiments, the substitution is aspartate (D) to asparagine(N); or aspartate (D) to histidine (H); or aspartate (D) to isoleucine(I); or aspartate (D) to threonine (T); or aspartate (D) to serine (S);or aspartate (D) to tyrosine (Y); or aspartate (D) to arginine (R); oraspartate (D) to leucine (L); or aspartate (D) to glutamine (Q); oraspartate (D) to glutamate (G). In another embodiment, the variant BCCPhas SEQ ID NO: 6 encompassing a polypeptide with a mutation thatincludes a substitution of aspartate (D) to asparagine (N). In anotherembodiment, the variant BCCP has SEQ ID NO: 4 o SEQ ID NO: 8encompassing a polypeptide with a mutation that includes a substitutionof aspartate (D) to histidine (H). In another embodiment, the variantBCCP has SEQ ID NO: 10 o SEQ ID NO: 12 encompassing a polypeptide with amutation that includes a substitution of aspartate (D) to isoleucine(I). In one embodiment, the variant BCCP has at least one mutation inits amino acid sequence and confers to a recombinant cell an increasedproduction of a malonyl-CoA-derived compound when compared to acorresponding wild type cell. In another embodiment, the variant BCCPhas at least one mutation in its amino acid sequence and may conferimproved acetyl-CoA carboxylase (ACC) activity to a recombinant cell,resulting in increased production of a malonyl-CoA-derived compound whencompared to a corresponding wild type cell. In another embodiment, thecell is a recombinant microorganism or recombinant host cell that can becontrasted with or compared to a wild type microorganism or wild typehost cell. In another embodiment, the cell is microbial in nature.

Yet another aspect of the disclosure provides a method of producing amalonyl-CoA-derived compound, including culturing a cell that expressesa variant BCCP in a fermentation broth containing a carbon source. Themalonyl-CoA-derived compound includes a fatty acid derivative, such as,for example, a fatty acid, a fatty acid methyl ester (FAME), a fattyacid ethyl ester (FAEE), a fatty alcohol, a fatty amine, a beta hydroxyfatty acid derivative, a bifunctional fatty acid derivative (e.g.,w-hydroxy fatty acids, w-hydroxy diols, w-hydroxy FAME, w-hydroxy FAEE),an unsaturated fatty acid derivative, as well as a non-fatty acid basedcompound such as a flavanone and/or a flavonoid, a polyketide, and3-hydroxypropionic acid. In one embodiment, the cell is a recombinantmicroorganism or recombinant host cell that can be contrasted with orcompared to a wild type microorganism or wild type host cell,respectively. In another embodiment, the cell is microbial in nature.

The disclosure further contemplates a variant operon controlling theexpression of a BCCP. In one embodiment, the operon results in a changein the BCCP expression in a recombinant cell as compared to a wild typecell. In one embodiment, the cell is a recombinant microbial host cellor recombinant microorganism as compared to a wild type microbial hostcell or wild type microorganism, respectively. In another embodiment,the operon results in an increase in the BCCP expression in arecombinant cell and thereby improves acetyl-CoA carboxylase (ACC)activity in the recombinant cell, resulting in increased production of amalonyl-CoA-derived compound when compared to a corresponding wild typecell. In one aspect, the variant operon further includes a promoter. Thepromoter includes, but is not limited to, a heterologous promoter, aheterologous promoter variant, and a synthetic promoter. In oneembodiment, the promoter includes a genetically modified accBC promoter,a naturally occurring E. coli promoter, or an E. coli promoter variant.In another embodiment, the promoter is an accBC promoter variant. Inanother embodiment, the promoter is a T5 promoter or T5 promotervariant. In one embodiment, the promoter is an accBC T5 promoter. Inanother embodiment, the accBC T5 promoter is selected from SEQ ID NOS:93, 94, 95, or 96 or variations thereof.

The disclosure further provides a recombinant microorganism or host cellencompassing a variant operon that controls the expression of a BCCP. Inone embodiment, the operon results in a change in the BCCP expression.In one embodiment, the operon results in an increase in the BCCPexpression in a recombinant cell, and thereby improves acetyl-CoAcarboxylase (ACC) activity in the recombinant cell, resulting inincreased production of a malonyl-CoA-derived compound when compared toa corresponding wild type cell. In another embodiment, the variantoperon further includes a promoter.

Another aspect of the disclosure provides a method of producing amalonyl-CoA-derived compound, including culturing a microorganism orhost cell that expresses a variant operon in a fermentation brothcontaining a carbon source. In one embodiment, the cell is a recombinantmicroorganism or recombinant host cell that can be contrasted with orcompared to a wild type microorganism or wild type host cell,respectively. In another embodiment, the cell is microbial in nature.The malonyl-CoA-derived compound includes a fatty acid, a fatty acidmethyl ester (FAME), a fatty acid ethyl ester (FAEE), a fatty alcohol, afatty amine, a beta hydroxy fatty acid derivative, a bifunctional fattyacid derivative (e.g., ω-hydroxy fatty acids, ω-hydroxy diols, ω-hydroxyFAME, ω-hydroxy FAEE), an unsaturated fatty acid derivative, as well asa non-fatty acid based compound such as a flavanone and/or a flavonoid,a polyketide, and 3-hydroxypropionic acid.

Still another aspect of the disclosure provides a method of producing amalonyl-CoA-derived compound, including culturing a host cell expressinga variant BCCP and a variant operon in a fermentation broth, containinga carbon source. In one embodiment, the cell is a recombinantmicroorganism or recombinant host cell that can be contrasted with orcompared to a wild type microorganism or wild type host cell,respectively. In another embodiment, the cell is microbial in nature.The malonyl-CoA-derived compound includes a fatty acid, a fatty acidmethyl ester (FAME), a fatty acid ethyl ester (FAEE), a fatty alcohol, afatty amine, a beta hydroxy fatty acid derivative, a bifunctional fattyacid derivative (including ω-hydroxy fatty acids, ω-hydroxy diols,ω-hydroxy FAME, ω-hydroxy FAEE), an unsaturated fatty acid derivative,as well as a non-fatty acid based compound such as a flavanone and/or aflavonoid, a polyketide, and 3-hydroxypropionic acid.

The disclosure further contemplates a microorganism that encompasses avariant biotin carboxyl carrier protein (BCCP) having at least onemutation in its amino acid sequence. In one embodiment, the variant BCCPhas a mutation in an N-terminal amino acid region. In anotherembodiment, the mutation is a substitution. In various embodiments, thesubstitution is aspartate (D) to asparagine (N); or aspartate (D) tohistidine (H); or aspartate (D) to isoleucine (I); or aspartate (D) tothreonine (T); or aspartate (D) to serine (S); or aspartate (D) totyrosine (Y); or aspartate (D) to arginine (R); or aspartate (D) toleucine (L); or aspartate (D) to glutamine (Q); or aspartate (D) toglutamate (G). In another embodiment, the variant BCCP has one or moremutation(s), including substitutions of aspartate (D) to asparagine (N);aspartate (D) to histidine (H); aspartate (D) to isoleucine (I);aspartate (D) to threonine (T); aspartate (D) to serine (S); aspartate(D) to tyrosine (Y); aspartate (D) to arginine (R); aspartate (D) toleucine (L); aspartate (D) to glutamine (Q); and/or aspartate (D) toglutamate (G). In another embodiment, the variant BCCP has SEQ ID NO: 6encompassing a polypeptide with a mutation that includes a substitutionof aspartate (D) to asparagine (N). In another embodiment, the variantBCCP has SEQ ID NO: 4 o SEQ ID NO: 8 encompassing a polypeptide with amutation that includes a substitution of aspartate (D) to histidine (H).In another embodiment, the variant BCCP has SEQ ID NO: 10 o SEQ ID NO:12 encompassing a polypeptide with a mutation that includes asubstitution of aspartate (D) to isoleucine (I). In another embodiment,the expression of the variant BCCP confers an increased production of amalonyl-CoA-derived compound to the microorganism. In anotherembodiment, the expression of the variant BCCP may confer improvedacetyl-CoA carboxylase (ACC) activity in the microorganism, resulting inincreased production of a malonyl-CoA-derived compound by themicroorganism. The malonyl-CoA-derived compound includes, but is notlimited to, a fatty acid, a fatty acid methyl ester (FAME), a fatty acidethyl ester (FAEE), a fatty alcohol, a fatty amine, a beta hydroxy fattyacid derivative, a bifunctional fatty acid derivative, an unsaturatedfatty acid derivative, a flavanone, a flavonoid, a polyketide, and3-hydroxypropionic acid. In one embodiment, the malonyl-CoA-derivedcompound is a FAME or a FAEE. In another embodiment, themalonyl-CoA-derived compound is a fatty alcohol. In another embodiment,the microorganism is a microbial cell. In yet another embodiment, themicrobial cell is a recombinant cell. Examples of microbial cellsinclude, but are not limited to, cells from the genus Escherichia,Bacillus, Cyanophyta, Lactobacillus, Zymomonas, Rhodococcus,Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola,Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium,Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces,Stenotrophamonas, Schizosaccharomyces, Yarrowia, and Streptomyces. Inone embodiment, the microbial cell is from the genus Escherichia. In oneembodiment, the microbial cell is from Escherichia coli. In anotherembodiment, the microbial cell is from a cyanobacteria or the genusCyanophyta. In another embodiment, the microbial cell is from acyanobacteria or Cyanophyta including, but not limited to,Prochlorococcus, Synechococcus, Synechocystis, Cyanothece, and Nostocpunctiforme. In another embodiment, the microbial cell is from aspecific cyanobacterial species including, but not limited to,Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, andSynechococcus sp. PCC7001.

Another aspect of the disclosure provides a recombinant microorganismhaving an altered expression of a nucleic acid sequence including accBor accC or a combination thereof, resulting in altered production of amalonyl-CoA-derived compound by the microorganism. In one embodiment,the altered expression is increased expression. In another embodiment,the altered expression is decreased expression. In yet anotherembodiment, the altered expression is due to a change in one or morepromoters that drive expression of the nucleic acid sequence. Thenucleic acid sequence of accB codes for BCCP. In one embodiment, thevariant nucleic acid sequence of accB codes for the variant BCCP. In oneembodiment, the malonyl-CoA-derived compound includes, but is notlimited to, a fatty acid, a fatty acid methyl ester (FAME), a fatty acidethyl ester (FAEE), a fatty alcohol, a fatty amine, a beta hydroxy fattyacid derivative, a bifunctional fatty acid derivative, an unsaturatedfatty acid derivative, a flavanone, a flavonoid, a polyketide, and3-hydroxypropionic acid. In one embodiment, the microorganism includes,but is not limited to, microorganisms from the genus Escherichia,Bacillus, Cyanophyta, Lactobacillus, Zymomonas, Rhodococcus,Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola,Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium,Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces,Stenotrophamonas, Schizosaccharomyces, Yarrowia, or Streptomyces. In oneembodiment, the microbial cell is from the genus Escherichia. In oneembodiment, the microbial cell is from Escherichia coli. In anotherembodiment, the microbial cell is from a cyanobacteria or the genusCyanophyta. In still another embodiment, the microorganism is acyanobacteria or Cyanophyta from Prochlorococcus, Synechococcus,Synechocystis, Cyanothece, or Nostoc punctiforme. In one embodiment, themicroorganism is a cyanobacterial species from Synechococcus elongatusPCC7942, Synechocystis sp. PCC6803, or Synechococcus sp. PCC7001.

Another aspect of the disclosure provides a microorganism or host cellthat has an altered expression of an ACC variant and further expresses afatty acid biosynthesis protein. In one embodiment, the host cell is amicrobial cell. In another embodiment, the host cell is a recombinantcell. In yet another embodiment, the host cell is a recombinantbacterial cell. In another embodiment, the ACC variant is a biotincarboxyl carrier protein (BCCP) or a biotin carboxylase (BC) or acombination thereof. In one embodiment, the altered expression isincreased or decreased expression. In one embodiment, the alteredexpression is increased expression, wherein the increased expressionresults in an increased production of a malonyl-CoA-derived compoundwhen the microbial cell is cultured with a carbon source.

In certain embodiments of the present disclosure the host cell mayfurther express a biosynthetic protein that has enzymatic activity thatcan increase production of fatty acid derivatives. In one embodiment,the protein with enzymatic activity may be natively present in the hostcell and its gene may be overexpressed via a promoter or other geneticalteration. In another embodiment, the protein with enzymatic activitymay be the result of an exogenous or heterologous gene that is expressedin the host cell. Examples of such enzymatic activities include, but arenot limited to, thioesterase activity (E.C. 3.1.2.* or E.C. 3.1.2.14 orE.C. 3.1.1.5), ester synthase activity (E.C. 2.3.1.75), acyl-ACPreductase (AAR) activity (E.C. 1.2.1.80), alcohol dehydrogenase activity(E.C. 1.1.1.1.), fatty alcohol acyl-CoA reductase (FAR) activity (E.C.1.1.1.*), carboxylic acid reductase (CAR) activity (EC 1.2.99.6),decarbonylase or deformylase activity, acyl-CoA reductase activity (E.C.1.2.1.50), acyl-CoA synthase (FadD) activity (E.C. 2.3.1.86), OleAactivity, and OleBCD activity.

In still another aspect, the disclosure provides a microorganism or hostcell that has an altered expression of an ACC variant and furtherexpresses a fatty acid biosynthesis protein, wherein the alteredexpression is increased expression that results in an increasedproduction of a malonyl-CoA-derived compound when the cell is culturedwith a carbon source. In one embodiment, the host cell is a microbialcell. In another embodiment, the host cell is a recombinant cell. In yetanother embodiment, the host cell is a recombinant bacterial cell. Instill another embodiment, the microorganism or host cell is arecombinant cell that can be compared to or contrasted with a wild typecell under the same conditions. Herein the malonyl-CoA-derived compoundincludes, but is not limited to, a fatty acid, a fatty acid methyl ester(FAME), a fatty acid ethyl ester (FAEE), a fatty alcohol, a fatty amine,a beta hydroxy fatty acid derivative, a bifunctional fatty acidderivative, an unsaturated fatty acid derivative, a flavanone, aflavonoid, a polyketide, and 3-hydroxypropionic acid. In one embodiment,the microbial cell is selected from cells of the genus Escherichia,Bacillus, Cyanophyta, Lactobacillus, Zymomonas, Rhodococcus,Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola,Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium,Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces,Stenotrophamonas, Schizosaccharomyces, Yarrowia, or Streptomyces.

Another aspect of the disclosure provides a variant biotin carboxylcarrier protein (BCCP) having SEQ ID NO: 6. In one embodiment, themutation is in an N-terminal amino acid region with a substitution ofaspartate (D) to asparagine (N), wherein the substitution is in aminoacid position 2. In another embodiment, the varian BCCP is encoded by avariant accB gene, wherein the variant accB gene has a nucleic acidsequence of SEQ ID NO: 5. Another aspect of the disclosure provides avariant biotin carboxyl carrier protein (BCCP) having SEQ ID NO: 4 orSEQ ID NO: 8. In one embodiment, the mutation is in an N-terminal aminoacid region with a substitution of aspartate (D) to histidine (H),wherein the substitution is in amino acid position 2. In anotherembodiment, the varian BCCP is encoded by a variant accB gene, whereinthe variant accB gene has a nucleic acid sequence of SEQ ID NO: 3 or SEQID NO: 7, respectively. Another aspect of the disclosure provides avariant biotin carboxyl carrier protein (BCCP) having SEQ ID NO: 10 orSEQ ID NO: 12. In one embodiment, the mutation is in an N-terminal aminoacid region with a substitution of aspartate (D) to isoleucine (I),wherein the substitution is in amino acid position 2. In anotherembodiment, the variant BCCP is encoded by a variant accB gene, whereinthe variant accB gene has a nucleic acid sequence of SEQ ID NO: 9 or SEQID NO: 11, respectively.

In various embodiments, the variant BCCPs confer to a recombinant cellan increased production of a malonyl-CoA-derived compound when comparedto a corresponding wild type cell, wherein said malonyl-CoA-derivedcompound includes a fatty acid derivative of a fatty acid, a fatty acidmethyl ester (FAME), a fatty acid ethyl ester (FAEE), a fatty alcohol, afatty amine, a beta hydroxy fatty acid derivative, a bifunctional fattyacid derivative, and an unsaturated fatty acid derivative; or anon-fatty acid compound such as a flavanone, a flavonoid, a polyketide,and 3-hydroxypropionic acid. The present disclosure further encompassesrecombinant microorganisms that include or express the variant BCCPs. Inone embodiment, the microorganisms are selected from microorganisms ofthe genus Escherichia, Bacillus, Cyanophyta, Lactobacillus, Zymomonas,Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora,Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor,Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes,Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces,Yarrowia, or Streptomyces.

Further contemplated is a method of producing a malonyl-CoA-derivedcompound, including culturing the recombinant microorganism expressingthe variant BCCP in a fermentation broth containing a carbon source. Themalonyl-derived compound produced by this method includes a fatty acidderivative including a fatty acid, a fatty acid methyl ester (FAME), afatty acid ethyl ester (FAEE), a fatty alcohol, a fatty amine, a betahydroxy fatty acid derivative, a bifunctional fatty acid derivative, andan unsaturated fatty acid derivative; or a non-fatty acid compound suchas a flavanone, a flavonoid, a polyketide, and 3-hydroxypropionic acid.

BRIEF DESCRIPTION OF THE FIGURES

The present disclosure is best understood when read in conjunction withthe accompanying figures, which serve to illustrate the preferredembodiments. It is understood, however, that the disclosure is notlimited to the specific embodiments disclosed in the figures.

FIG. 1 is a schematic of one embodiment of an engineered biochemicalpathway that involves acetyl-CoA carboxylase (ACC) variants such as avariant biotin carboxyl carrier protein (BCCP). As depicted, BCCP mayconfer improved acetyl-CoA carboxylase (ACC) activity when expressed ina cell. This may lead to increased production of malonyl-CoA andacyl-ACP, which in turn may lead to increased production ofmalonyl-CoA-derived compounds, including, for example, fatty acidderivatives such as fatty esters, fatty aldehydes, fatty alcohols, fattyacids, and other fatty acid derivatives.

FIG. 2 depicts an alignment of seven amino acid sequences of BCCP fromseven different species. The boxed area shows a motif that is conservedacross most BCCP species.

FIG. 3 is a schematic of another embodiment of an engineered biochemicalpathway that involves acetyl-CoA carboxylase (ACC) variants such as avariant biotin carboxyl carrier protein (BCCP). As depicted, BCCP mayconfer improved acetyl-CoA carboxylase (ACC) activity when expressed ina cell. This may lead to increased production of malonyl-CoA andacyl-CoA, which in turn may lead to increased production ofmalonyl-CoA-derived compounds, including, for example, fatty acidderivatives such as fatty esters, fatty aldehydes, fatty alcohols, fattyacids, and other fatty acid derivatives.

FIG. 4 is a summary of several embodiments of an engineered biochemicalpathway that involves acetyl-CoA carboxylase (ACC) variants such as avariant biotin carboxyl carrier protein (BCCP). As shown, BCCP mayconfer improved acetyl-CoA carboxylase (ACC) activity when expressed ina cell. This may lead to increased production of malonyl-CoA andmalonyl-CoA derived compounds, including polyketides, 3-hydroxypropionicacid (3-HP), flavanones and flavonoids as well as increasedintermediates such as, for example, increased acyl-CoA (see also FIG.3); increased acyl-ACP (see also FIG. 1); as well as increased malonate(or malonic acid). Increased intermediates may further lead to increasedend-products such as fatty acid derivatives, including fatty acids,fatty esters, fatty aldehydes, fatty alcohols and other fatty acidderivatives.

FIG. 5 shows a graph that depicts the FAS titer (FAME) as a result ofexpressing various BCCP variants (at position 2 of the accB gene) in E.coli host cells. WT is the control for the wild-type ACC complex. Someof these BCCP variants improved FAS titer over 5-fold. (see also Table1).

DETAILED DESCRIPTION

General Overview

The disclosure relates to variant acetyl-CoA carboxylase (ACC)polypeptide(s) or ACC variant(s) that can be expressed in amicroorganism. These ACC variants are genetically altered and arebelieved to confer improved enzymatic activity for the increasedproduction of malonyl-CoA derived compounds including fatty acidderivatives. Herein, the disclosure relates to polypeptide(s) andprotein(s) that may lead to improved acetyl-CoA carboxylase (ACC)activity when expressed in a host cell, when compared to thecorresponding ACC activity in a wild type cell. In order to illustratethis, ACC genes were altered by introducing mutations in one ACC gene aswell as one ACC operon. Both of these alterations can independentlyincrease fatty acid derivative production in a host cell. Thesemutations are expected to improve the titer and yield of a productderived from malonyl-CoA, including but not limited to, fattyacid-derived compounds (i.e., fatty acid derivatives) such as, forexample, fatty acids, fatty esters, fatty alcohols, fatty aldehydes,fatty amines, bifunctional fatty acid derivatives, and non-fatty acidbased compounds such as, for example, flavanones and flavonoids,polyketides, and 3-hydroxypropionic acid. Examples of fatty esters arefatty acid methyl esters (FAME) and fatty acid ethyl esters (FAEE).Examples of bifunctional fatty acid derivatives include, but are notlimited to, ω-hydroxy fatty acids, ω-hydroxy diols, ω-hydroxy FAME, andω-hydroxy FAEE.

It has been stipulated, that in order to produce higher yields ofmalonyl-CoA derived compounds, the increased expression of all four ACCgenes encoding the complete ACC complex is required (Davis et al. (2000)supra). However, the present disclosure reveals the surprising findingthat targeted mutations in only one ACC gene can improve the productionof compounds that are derived from malonyl-CoA, including fatty acidderivatives. For example, targeted mutations in the accB gene and/ortargeted expression changes in the accBC operon significantly improvedfatty ester production by up to 630 percent (see FIG. 5 as well as Table1 and the Examples, infra). Without wishing to be bound by theory, thevariant ACC polypeptides or ACC variants are believed to directly orindirectly confer onto ACC complexes improved enzymatic activity thatleads to a higher production of malonyl-CoA derived compounds in hostcells. The specific activity of the host cell is believed to beincreased, thereby resulting in increased production of malonyl-CoAderived compounds. Such malonyl-CoA derived compounds include fatty acidderivatives and non-fatty acid based compounds.

Definitions

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a hostcell” includes two or more such host cells, reference to “a fatty ester”includes one or more fatty esters, or mixtures of esters, reference to“a nucleic acid sequence” includes one or more nucleic acid sequences,reference to “an enzyme” includes one or more enzymes, and the like.

Sequence Accession numbers throughout this description were obtainedfrom databases provided by the NCBI (National Center for BiotechnologyInformation) maintained by the National Institutes of Health, U.S.A.(which are identified herein as “NCBI Accession Numbers” oralternatively as “GenBank Accession Numbers” or alternatively a simply“Accession Numbers”), and from the UniProt Knowledgebase (UniProtKB) andSwiss-Prot databases provided by the Swiss Institute of Bioinformatics(which are identified herein as “UniProtKB Accession Numbers”).

Enzyme Classification (EC) numbers are established by the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (IUBMB), description of which is available on the IUBMB EnzymeNomenclature website on the World Wide Web. EC numbers classify enzymesaccording to the reaction they catalyze. For example, the acetyl-CoAcarboxylase (ACC) enzymatic activity is classified under E.C. 6.4.1.2.ACC is a multi-subunit enzyme complex present in most prokaryotes and inthe chloroplasts of most plants and algae. ACC catalyzes the reaction ofATP and acetyl-CoA and HCO₃— to ADP and phosphate and malonyl-CoA. Thefunctionality of ACC is conserved in most prokaryotes from one speciesto the next. Thus, different microbial species can carry out the sameacetyl-CoA carboxylase (ACC) enzymatic activity that is classified underE.C. 6.4.1.2.

As used herein, the term “nucleotide” refers to a monomeric unit of apolynucleotide that consists of a heterocyclic base, a sugar, and one ormore phosphate groups. The naturally occurring bases (guanine, (G),adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typicallyderivatives of purine or pyrimidine, though it should be understood thatnaturally and non-naturally occurring base analogs are also included.The naturally occurring sugar is the pentose (five-carbon sugar)deoxyribose (which forms DNA) or ribose (which forms RNA), though itshould be understood that naturally and non-naturally occurring sugaranalogs are also included. Nucleic acids are typically linked viaphosphate bonds to form nucleic acids or polynucleotides, though manyother linkages are known in the art (e.g., phosphorothioates,boranophosphates, and the like).

The term “polynucleotide” refers to a polymer of ribonucleotides (RNA)or deoxyribonucleotides (DNA), which can be single-stranded ordouble-stranded and which can contain non-natural or alterednucleotides. The terms “polynucleotide,” “nucleic acid sequence,” and“nucleotide sequence” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, either RNA or DNA. Theseterms refer to the primary structure of the molecule, and thus includedouble- and single-stranded DNA, and double- and single-stranded RNA.The terms include, as equivalents, analogs of either RNA or DNA madefrom nucleotide analogs and modified polynucleotides such as, though notlimited to methylated and/or capped polynucleotides. The polynucleotidecan be in any form, including but not limited to, plasmid, viral,chromosomal, EST, cDNA, mRNA, and rRNA.

As used herein, the terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The term“recombinant polypeptide” refers to a polypeptide that is produced byrecombinant techniques, wherein generally DNA or RNA encoding theexpressed protein is inserted into a suitable expression vector that isin turn used to transform a host cell to produce the polypeptide. DNA orRNA encoding the expressed protein can also be inserted into the hostchromosome via homologous recombination or other means well known in theart, and is so used to transform a host cell to produce the polypeptide.Similarly, the terms “recombinant polynucleotide” or “recombinantnucleic acid” or “recombinant DNA” are produced by recombinanttechniques that are known to those of skill in the art.

As used herein, the terms “homolog,” and “homologous” refer to apolynucleotide or a polypeptide comprising a sequence that is at leastabout 50 percent (%) identical to the corresponding polynucleotide orpolypeptide sequence. Preferably homologous polynucleotides orpolypeptides have polynucleotide sequences or amino acid sequences thathave at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99%homology to the corresponding amino acid sequence or polynucleotidesequence. As used herein the terms sequence “homology” and sequence“identity” are used interchangeably.

One of ordinary skill in the art is well aware of methods to determinehomology between two or more sequences. Briefly, calculations of“homology” between two sequences can be performed as follows. Thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In one preferred embodiment, thelength of a first sequence that is aligned for comparison purposes is atleast about 30%, preferably at least about 40%, more preferably at leastabout 50%, even more preferably at least about 60%, and even morepreferably at least about 70%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, or about100% of the length of a second sequence. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions of the first and second sequences are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position. The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps and the length of each gap, that need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent homology between two sequences can beaccomplished using a mathematical algorithm, such as BLAST (Altschul etal. (1990) J Mol. Biol. 215(3):403-410). The percent homology betweentwo amino acid sequences also can be determined using the Needleman andWunsch algorithm that has been incorporated into the GAP program in theGCG software package, using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch (1970) J Mol. Biol.48:444-453). The percent homology between two nucleotide sequences alsocan be determined using the GAP program in the GCG software package,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. One of ordinary skill in theart can perform initial homology calculations and adjust the algorithmparameters accordingly. A preferred set of parameters (and the one thatshould be used if a practitioner is uncertain about which parametersshould be applied to determine if a molecule is within a homologylimitation of the claims) are a Blossum 62 scoring matrix with a gappenalty of 12, a gap extend penalty of 4, and a frameshift gap penaltyof 5. Additional methods of sequence alignment are known in thebiotechnology arts (see, e.g., Rosenberg (2005) BMC Bioinformatics6:278; Altschul et al. (2005) FEBS J. 272(20):5101-5109).

The term “hybridizes under low stringency, medium stringency, highstringency, or very high stringency conditions” describes conditions forhybridization and washing. Guidance for performing hybridizationreactions can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methodsare described in that reference and either method can be used. Specifichybridization conditions referred to herein are as follows: (1) lowstringency hybridization conditions—6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridizationconditions—6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (3) high stringency hybridizationconditions—6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and (4) very high stringency hybridizationconditions—0.5M sodium phosphate, 7% SDS at 65° C., followed by one ormore washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions(4) are the preferred conditions unless otherwise specified.

An “endogenous” polypeptide refers to a polypeptide encoded by thegenome of the parental cell (or host cell). An “exogenous” polypeptiderefers to a polypeptide which is not encoded by the genome of theparental cell. A variant or mutant polypeptide is an example of anexogenous polypeptide. Thus, a non-naturally-occurring nucleic acidmolecule is considered to be exogenous to a cell once introduced intothe cell. A nucleic acid molecule that is naturally-occurring can alsobe exogenous to a particular cell. For example, an entire codingsequence isolated from cell X is an exogenous nucleic acid with respectto cell Y once that coding sequence is introduced into cell Y, even if Xand Y are the same cell type.

The term “overexpressed” means that a gene is caused to be transcribedat an elevated rate compared to the endogenous transcription rate forthat gene. In some examples, overexpression additionally includes anelevated rate of translation of the gene compared to the endogenoustranslation rate for that gene. Methods of testing for overexpressionare well known in the art, for example transcribed RNA levels can beassessed using rtPCR and protein levels can be assessed using SDS pagegel analysis.

The term “heterologous” means derived from a different organism,different cell type, or different species. As used herein it refers to anucleotide-, polynucleotide-, polypeptide- or protein sequence, notnaturally present in a given organism. For example, a polynucleotidesequence that is native to cyanobacteria can be introduced into a hostcell of E. coli by recombinant methods, and the polynucleotide fromcyanobacteria is then heterologous to the E. coli cell (e.g.,recombinant cell). The term “heterologous” may also be used withreference to a nucleotide-, polynucleotide-, polypeptide-, or proteinsequence which is present in a recombinant host cell in a non-nativestate. For example, a “heterologous” nucleotide, polynucleotide,polypeptide or protein sequence may be modified relative to the wildtype sequence naturally present in the corresponding wild type hostcell, e.g., a modification in the level of expression or in the sequenceof a nucleotide, polynucleotide, polypeptide or protein.

As used herein, the term “fragment” of a polypeptide refers to a shorterportion of a full-length polypeptide or protein ranging in size from twoamino acid residues to the entire amino acid sequence minus one aminoacid residue. In certain embodiments of the disclosure, a fragmentrefers to the entire amino acid sequence of a domain of a polypeptide orprotein (e.g., a substrate binding domain or a catalytic domain).

The term “mutagenesis” refers to a process by which the geneticinformation of an organism is changed in a stable manner. Mutagenesis ofa protein coding nucleic acid sequence produces a mutant protein.Mutagenesis also refers to changes in non-coding nucleic acid sequencesthat result in modified protein activity.

A “mutation”, as used herein, refers to a permanent change in a nucleicacid position of a gene or in an amino acid position of a polypeptide orprotein. Mutations include substitutions, additions, insertions, anddeletions. For example, a mutation in an amino acid position can be asubstitution of one type of amino acid with another type of amino acid(e.g., an aspartate (D) may be substituted with an tyrosine (Y); alysine (L) may be substituted with a threonine (T); etc.). As such, apolypeptide or a protein can have one or more mutations wherein oneamino acid is substituted with another amino acid. For example, an ACCrelated polypeptide or protein can have one or more mutations in itsamino acid sequence.

As used herein, the term “gene” refers to nucleic acid sequencesencoding either an RNA product or a protein product, as well asoperably-linked nucleic acid sequences affecting the expression of theRNA or protein (e.g., such sequences include but are not limited topromoter or enhancer sequences) or operably-linked nucleic acidsequences encoding sequences that affect the expression of the RNA orprotein (e.g., such sequences include but are not limited to ribosomebinding sites or translational control sequences).

Expression control sequences are known in the art and include, forexample, promoters, enhancers, polyadenylation signals, transcriptionterminators, internal ribosome entry sites (IRES), and the like, thatprovide for the expression of the polynucleotide sequence in a hostcell. Expression control sequences interact specifically with cellularproteins involved in transcription (Maniatis et al., Science, 236:1237-1245 (1987)). Exemplary expression control sequences are describedin, for example, Goeddel, Gene Expression Technology: Methods inEnzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). In themethods of the disclosure, an expression control sequence is operablylinked to a polynucleotide sequence. By “operably linked” is meant thata polynucleotide sequence and an expression control sequence(s) areconnected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins) arebound to the expression control sequence(s). Operably linked promotersare located upstream of the selected polynucleotide sequence in terms ofthe direction of transcription and translation. Operably linkedenhancers can be located upstream, within, or downstream of the selectedpolynucleotide.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid, i.e., a polynucleotidesequence, to which it has been linked. One type of useful vector is anepisome (i.e., a nucleic acid capable of extra-chromosomal replication).Useful vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked. Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors.” In general, expressionvectors of utility in recombinant DNA techniques are often in the formof “plasmids,” which refer generally to circular double stranded DNAloops that, in their vector form, are not bound to the chromosome. Theterms “plasmid” and “vector” are used interchangeably herein, in as muchas a plasmid is the most commonly used form of vector. However, alsoincluded are such other forms of expression vectors that serveequivalent functions and that become known in the art subsequentlyhereto. In some embodiments, a recombinant vector further includes apromoter operably linked to the polynucleotide sequence. In someembodiments, the promoter is a developmentally-regulated, anorganelle-specific, a tissue-specific, an inducible, a constitutive, ora cell-specific promoter. The recombinant vector typically comprises atleast one sequence selected from an expression control sequenceoperatively coupled to the polynucleotide sequence; a selection markeroperatively coupled to the polynucleotide sequence; a marker sequenceoperatively coupled to the polynucleotide sequence; a purificationmoiety operatively coupled to the polynucleotide sequence; a secretionsequence operatively coupled to the polynucleotide sequence; and atargeting sequence operatively coupled to the polynucleotide sequence.In certain embodiments, the nucleotide sequence is stably incorporatedinto the genomic DNA of the host cell, and the expression of thenucleotide sequence is under the control of a regulated promoter region.The expression vectors described herein include a polynucleotidesequence described herein in a form suitable for expression of thepolynucleotide sequence in a host cell. It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of polypeptide desired, etc. The expression vectorsdescribed herein can be introduced into host cells to producepolypeptides, including fusion polypeptides, encoded by thepolynucleotide sequences as described herein.

The terms “recombinant cell” and “recombinant host cell” are usedinterchangeably herein and refer to a cell that may express an ACCvariant and/or encompasses a variant operon that may increase thespecific activity of the recombinant cell to produce malonyl-CoA derivedcompounds. A recombinant cell can be derived from a microorganism suchas a bacterium, a virus or a fungus. In addition, a recombinant cell canbe derived from a plant or an animal cell. The recombinant cell can beused to produce one or more fatty acid derivatives including, but notlimited to, fatty acids, fatty esters (e.g., waxes, fatty acid esters,fatty esters, fatty acid methyl esters (FAME), fatty acid ethyl esters(FAEE)), fatty alcohols, short and long chain alcohols, fatty aldehydes,hydrocarbons, fatty amines, terminal olefins, internal olefins, ketones,bifunctional fatty acid derivatives (e.g., ω-hydroxy fatty acids,ω-hydroxy diols, ω-hydroxy FAME, ω-hydroxy FAEE); a well as non-fattyacid compounds such as flavanones, flavonoids, polyketides, and3-hydroxypropionic acid. In some embodiments, the recombinant cellincludes one or more polynucleotides, each polynucleotide encoding apolypeptide having fatty acid biosynthetic enzyme activity, wherein therecombinant cell produces a fatty acid derivative composition whencultured in the presence of a carbon source under conditions effectiveto express the polynucleotides.

As used herein, the term “microorganism” refers to a microscopicorganism. Examples of a microorganism are a bacterium, a virus, or afungus. In one embodiment, a microorganism is a bacterial cell. Inanother embodiment, a microorganism is a prokaryote or prokaryotic cell.In yet another embodiment, a microorganism is a fungal cell such as ayeast cell. In another embodiment, a microorganism is a viral cell. In arelated embodiment, a “recombinant microorganism” is a microorganismthat has been genetically altered and expresses or encompasses anexogenous and/or heterologous nucleic acid sequence.

As used herein “acyl-ACP” refers to an acyl thioester formed between thecarbonyl carbon of alkyl chain and the sulfhydryl group of thephosphopantetheinyl moiety of an acyl carrier protein (ACP). Thephosphopantetheinyl moiety is post-translationally attached to aconserved serine residue on the ACP by the action of holo-acyl carrierprotein synthase (ACPS), a phosphopantetheinyl transferase. In someembodiments an acyl-ACP is an intermediate in the synthesis of fullysaturated acyl-ACPs. In other embodiments an acyl-ACP is an intermediatein the synthesis of unsaturated acyl-ACPs. In some embodiments, thecarbon chain will have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons. Each of theseacyl-ACPs are substrates for enzymes that convert them to fatty acidderivatives.

The term “malonyl-CoA derived compound” includes any compound orchemical entity (i.e., intermediate or end product) that is made via abiochemical pathway, wherein malonyl-CoA functions as intermediateand/or is made upstream of the compound or chemical entity (e.g., seeFIG. 4). For example, a malonyl-CoA derived compound includes, but isnot limited to, a fatty acid derivative such as, for example, a fattyacid; a fatty ester including, but not limited to a fatty acid methylester (FAME) and/or a fatty acid ethyl ester (FAEE); a fatty alcohol; afatty aldehyde; a fatty amine; an alkane; an olefin or alkene; ahydrocarbon; a beta hydroxy fatty acid derivative, a bifunctional fattyacid derivative, and an unsaturated fatty acid derivative. A malonyl-CoAderived compound further includes, but is not limited to, a non-fattyacid compound such as, for example, a flavanone, a flavonoid, apolyketide, malonate, and 3-hydroxypropionic acid.

The term “fatty acid” means a carboxylic acid having the formula RCOOH.R represents an aliphatic group, preferably an alkyl group. R cancomprise between about 4 and about 22 carbon atoms. Fatty acids can havea branched chain or straight chain and may be saturated,monounsaturated, or polyunsaturated.

A “fatty acid derivative” is a product made in part from the fatty acidbiosynthetic pathway of the production host organism. “Fatty acidderivatives” include products made from malonyl-CoA derived compoundsincluding acyl-ACP or acyl-ACP derivatives. Exemplary fatty acidderivatives include fatty acids, fatty esters (e.g., waxes, fatty acidesters, fatty esters, fatty acid methyl esters (FAME), fatty acid ethylesters (FAEE)), fatty amines, fatty aldehydes, fatty alcohols, short andlong chain alcohols, hydrocarbons, ketones, terminal olefins, internalolefins, ketones, beta hydroxy fatty acid derivatives, bifunctionalfatty acid derivatives (e.g., ω-hydroxy fatty acids, ω-hydroxy diols,ω-hydroxy FAME, ω-OH FAEE), and unsaturated fatty acid derivatives.“Fatty acid derivatives” also include products made from malonyl-CoAderived compounds such as acyl-CoA or acyl-CoA derivatives.

A “fatty acid derivative composition” as referred to herein is producedby a recombinant host cell and typically includes a mixture of fattyacid derivatives. In some cases, the mixture includes more than one typeof fatty acid derivative product (e.g., fatty acids, fatty esters, fattyalcohols, fatty aldehydes, fatty amine, bifunctional fatty acidderivatives, etc.). In other cases, a fatty acid derivative compositionmay include, for example, a mixture of fatty esters (or another fattyacid derivative) with different chain lengths, saturation and/orbranching characteristics. In still other cases, the fatty acidderivative composition may comprise both a mixture of more than one typeof fatty acid derivative product and fatty acid derivatives withdifferent chain lengths, saturation and/or branching characteristics. Inyet other cases, a fatty acid derivative composition may include, forexample, a mixture of fatty esters and beta hydroxy esters. In stillother cases, a fatty acid derivative composition may include, forexample, a mixture of fatty alcohols and fatty aldehydes. In still othercases, a fatty acid derivative composition may include, for example, amixture of FAME and/or FAEE.

The terms “variant biotin carboxyl carrier protein (BCCP)” and “biotincarboxyl carrier protein (BCCP) variant” are used interchangeably hereinand refer to an ACC variant that has one or more mutations in its aminoacid sequence. In one example, the amino acid sequence ranges from 1(i.e., the initial methionine (M) based on the ATG start site) to 156.Such a BCCP variant can have one or more mutation(s) in the amino acidposition 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, and/or 156. In one embodiment, themutations include mutations in the N-terminal amino acid region thatranges from about position 1 to about position 60. In one embodiment,the mutations include mutations in amino acid position 2 (right afterthe start codon).

The term “expression confers to (or results in) a recombinant cell withan increased production of a malonyl-CoA-derived compound when comparedto a corresponding wild type cell” refers to the function of an ACCrelated polypeptide or protein that has one or more mutations in itsamino acid sequence (i.e., an ACC variant or ACC mutant) and causes in acell improved production of malonyl-CoA derived compound(s) whenexpressed in that cell, when compared to a wild type cell that does notexpress the ACC variant or mutant. It also refers to the function of anACC variant that, when expressed in a cell, has the effect of causing ahigher specific activity of the cell in producing malonyl-CoA derivedcompound(s). Without wishing to be bound by theory, this may be theresult of directly or indirectly causing a higher acetyl-CoA carboxylase(ACC) enzymatic activity (E.C. 6.4.1.2) in the cell. This can bemeasured by comparing the titer and/or yield of a malonyl-CoA derivedcompound produced by the cell expressing the ACC variant with the titerand/or yield of a malonyl-CoA derived compound produced by acorresponding wild type cell (i.e., a cell that does not express the ACCvariant). Those of skill in the art will appreciate that the methods formeasurement are readily available, including, for example, gaschromatography flame ionization detector (GC-FID) and others. An exampleof an ACC variant protein is a biotin carboxyl carrier protein (BCCP)variant. The ACC variant(s) may encompass mutations in one and/or two ofany of the four subunits of the ACC complex. The ACC variant(s) mayencompass changes in concentration in any of one and/or two of the foursubunits. When a cell has been transformed with an ACC variant it is acell that expresses the ACC variant (e.g., a recombinant cell). In oneembodiment, the titer and/or yield of a malonyl-CoA derived compoundproduced by a cell that expresses the ACC variant is at least twice thatof a corresponding wild type cell (i.e., a corresponding cell that doesnot express the ACC variant). In another embodiment, the titer and/oryield of a malonyl-CoA derived compound produced by a cell thatexpresses the ACC variant is at least about 1 times, at least about 2times, at least about 3 times, at least about 4 times, at least about 5times, at least about 6 times, at least about 7 times, at least about 8times, at least about 9 times, or at least about 10 times greater thanthat of a corresponding wild type cell. In one embodiment, the titerand/or yield of a malonyl-CoA derived compound produced by a cellexpressing the ACC variant is at least about 1 percent, at least about 2percent, at least about 3 percent, at least about 4 percent, at leastabout 5 percent, at least about 6 percent, at least about 7 percent, atleast about 8 percent, at least about 9 percent, or about 10 percentgreater than that of a corresponding wild type cell. In anotherembodiment, the titer and/or yield due to the expression of an ACCvariant is at least about 20 percent to at least about 100 percentgreater than that of the wild type ACC complex. In one embodiment, thetiter and/or yield of a malonyl-CoA derived compound produced by a cellis at least about 20 percent, at least about 25 percent, at least about30 percent, at least about 35 percent, at least about 40 percent, atleast about 45 percent at least about 50 percent, at least about 55percent, at least about 60 percent, at least about 65 percent, at leastabout 70 percent, at least about 75 percent, at least about 80 percent,at least about 85 percent, at least about 90 percent, at least about 95percent, at least about 97 percent, at least about 98 percent, or atleast about 100 percent greater than that of the corresponding wild typecell. In another embodiment, the titer and/or yield of a malonyl-CoAderived compound produced by a cell is at least about 200 percent, atleast about 250 percent, at least about 300 percent, at least about 350percent, at least about 400 percent, at least about 450 percent at leastabout 500 percent, at least about 550 percent, at least about 600percent, at least about 610, 620, 630, 640 or 650 percent, at leastabout 700 percent, at least about 750 percent, at least about 800percent, or at least about 850 percent greater than that of thecorresponding wild type cell.

As used herein, the term “fatty acid biosynthetic pathway” means abiosynthetic pathway that produces fatty acid derivatives. The fattyacid biosynthetic pathway may include additional enzymes to producefatty acid derivatives having desired characteristics.

As used herein, “fatty ester” means an ester having the formula RCOOR′.A fatty ester as referred to herein can be any ester made from a fattyacid, for example a fatty acid ester. In some embodiments, the R groupis at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, or at least 19, carbons in length.Alternatively, or in addition, the R group is 20 or less, 19 or less, 18or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6or less carbons in length. Thus, the R group can have an R group boundedby any two of the above endpoints. For example, the R group can be 6-16carbons in length, 10-14 carbons in length, or 12-18 carbons in length.In some embodiments, the fatty ester composition comprises one or moreof a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19,C20, C21, C22, C23, C24, C25, and a C26 fatty ester. In otherembodiments, the fatty ester composition includes one or more of a C6,C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, and a C18 fattyester. In still other embodiments, the fatty ester composition includesC12, C14, C16 and C18 fatty esters; C12, C14 and C16 fatty esters; C14,C16 and C18 fatty esters; or C12 and C14 fatty esters.

The R group of a fatty acid derivative, for example a fatty ester, canbe a straight chain or a branched chain. Branched chains may have morethan one point of branching and may include cyclic branches. In someembodiments, the branched fatty acid, branched fatty aldehyde, orbranched fatty ester is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15,C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 branchedfatty acid, branched fatty aldehyde, or branched fatty ester. In certainembodiments, the branched fatty acid, branched fatty aldehyde, orbranched fatty ester is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15,C16, C17, or C18 branched fatty acid, or branched fatty ester. A fattyester of the present disclosure may be referred to as containing an Aside and a B side. As used herein, an “A side” of an ester refers to thecarbon chain attached to the carboxylate oxygen of the ester. As usedherein, a “B side” of an ester refers to the carbon chain comprising theparent carboxylate of the ester. When the fatty ester is derived fromthe fatty acid biosynthetic pathway, the A side is typically contributedby an alcohol, and the B side is contributed by a fatty acid.

Any alcohol can be used to form the A side of the fatty esters. Forexample, the alcohol can be derived from the fatty acid biosyntheticpathway, such as those describe herein. Alternatively, the alcohol canbe produced through non-fatty acid biosynthetic pathways. Moreover, thealcohol can be provided exogenously. For example, the alcohol can besupplied in the fermentation broth in cases where the fatty ester isproduced by an organism. Alternatively, a carboxylic acid, such as afatty acid or acetic acid, can be supplied exogenously in instanceswhere the fatty ester is produced by an organism that can also producealcohol.

The carbon chains comprising the A side or B side of the ester can be ofany length. In one embodiment, the A side of the ester is at least about1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. Whenthe fatty ester is a fatty acid methyl ester, the A side of the ester is1 carbon in length. When the fatty ester is a fatty acid ethyl ester,the A side of the ester is 2 carbons in length. The B side of the estercan be at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26carbons in length. The A side and/or the B side can be straight orbranched chain. The branched chains can have one or more points ofbranching. In addition, the branched chains can include cyclic branches.Furthermore, the A side and/or B side can be saturated or unsaturated.If unsaturated, the A side and/or B side can have one or more points ofunsaturation. In addition, the alcohol group of a fatty ester producedin accordance with the present disclosure need not be in the primary(C1) position. In one embodiment, the fatty ester is producedbiosynthetically. In this embodiment, first the fatty acid is“activated.” Non-limiting examples of “activated” fatty acids areacyl-CoA, acyl ACP, and acyl phosphate. Acyl-CoA can be a direct productof fatty acid biosynthesis or degradation. In addition, acyl-CoA can besynthesized from a free fatty acid, a CoA, and an adenosine nucleotidetriphosphate (ATP). An example of an enzyme which produces acyl-CoA isacyl-CoA synthase.

In certain embodiments, the branched fatty acid derivative is aniso-fatty acid derivative, for example an iso-fatty ester, or ananteiso-fatty acid derivative, e.g., an anteiso-fatty ester. Inexemplary embodiments, the branched fatty acid derivative is selectedfrom iso-C7:0, iso-C8:0, iso-C9:0, iso-C10:0, iso-C11:0, iso-C12:0,iso-C13:0, iso-C14:0, iso-C15:0, iso-C16:0, iso-C17:0, iso-C18:0,iso-C19:0, anteiso-C7:0, anteiso-C8:0, anteiso-C9:0, anteiso-C10:0,anteiso-C11:0, anteiso-C12:0, anteiso-C13:0, anteiso-C14:0,anteiso-C15:0, anteiso-C16:0, anteiso-C17:0, anteiso-C18:0, and ananteiso-C19:0 branched fatty ester.

The R group of a branched or unbranched fatty acid derivative can besaturated or unsaturated. If unsaturated, the R group can have one ormore than one point of unsaturation. In some embodiments, theunsaturated fatty acid derivative is a monounsaturated fatty acidderivative. In certain embodiments, the unsaturated fatty acidderivative is a C6:1, C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1,C14:1, C15:1, C16:1, C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1,C24:1, C25:1, or a C26:1 unsaturated fatty acid derivative. In certainembodiments, the unsaturated fatty acid derivative, is a C10:1, C12:1,C14:1, C16:1, or C18:1 unsaturated fatty acid derivative. In otherembodiments, the unsaturated fatty acid derivative is unsaturated at theomega-7 position. In certain embodiments, the unsaturated fatty acidderivative comprises a cis double bond.

As used herein, the term “clone” typically refers to a cell or group ofcells descended from and essentially genetically identical to a singlecommon ancestor, for example, the bacteria of a cloned bacterial colonyarose from a single bacterial cell.

As used herein, the term “culture” typical refers to a liquid mediacomprising viable cells. In one embodiment, a culture comprises cellsreproducing in a predetermined culture media under controlledconditions, for example, a culture of recombinant host cells grown inliquid media comprising a selected carbon source and nitrogen.“Culturing” or “cultivation” refers to growing a population of hostcells (e.g., recombinant host cells) under suitable conditions in aliquid or solid medium. In certain embodiments, culturing refers to thefermentative bioconversion of a substrate to an end-product. Culturingmedia are well known and individual components of such culture media areavailable from commercial sources, e.g., Difco™ media and BBL™ media. Inone non-limiting example, the aqueous nutrient medium is a “rich medium”including complex sources of nitrogen, salts, and carbon, such as YPmedium, comprising 10 g/L of peptone and 10 g/L yeast extract of such amedium.

As used herein, “modified” or an “altered level of” activity of aprotein, for example an enzyme, in a recombinant host cell refers to adifference in one or more characteristics in the activity determinedrelative to the parent or native host cell. Typically, differences inactivity are determined between a recombinant host cell, having modifiedactivity, and the corresponding wild-type host cell (e.g., comparison ofa culture of a recombinant host cell relative to the correspondingwild-type host cell). Modified activities can be the result of, forexample, modified amounts of protein expressed by a recombinant hostcell (e.g., as the result of increased or decreased number of copies ofDNA sequences encoding the protein, increased or decreased number ofmRNA transcripts encoding the protein, and/or increased or decreasedamounts of protein translation of the protein from mRNA); changes in thestructure of the protein (e.g., changes to the primary structure, suchas, changes to the protein's coding sequence that result in changes insubstrate specificity, changes in observed kinetic parameters); andchanges in protein stability (e.g., increased or decreased degradationof the protein). In some embodiments, the polypeptide is a mutant or avariant of any of the polypeptides described herein, e.g., a variant ACCincluding a variant BCCP. In certain instances, the coding sequences forthe polypeptides described herein are codon optimized for expression ina particular host cell. For example, for expression in E. coli, one ormore codons can be optimized (Grosjean can et al. (1982) Gene18:199-209).

The term “regulatory sequences” as used herein typically refers to asequence of bases in DNA, operably-linked to DNA sequences encoding aprotein that ultimately controls the expression of the protein. Examplesof regulatory sequences include, but are not limited to, RNA promotersequences, transcription factor binding sequences, transcriptiontermination sequences, modulators of transcription (such as enhancerelements), nucleotide sequences that affect RNA stability, andtranslational regulatory sequences (such as, ribosome binding sites(e.g., Shine-Dalgarno sequences in prokaryotes or Kozak sequences ineukaryotes), initiation codons, termination codons). As used herein, thephrase “the expression of said nucleotide sequence is modified relativeto the wild type nucleotide sequence,” means an increase or decrease inthe level of expression and/or activity of an endogenous nucleotidesequence or the expression and/or activity of a heterologous ornon-native polypeptide-encoding nucleotide sequence. The terms “alteredlevel of expression” and “modified level of expression” are usedinterchangeably and mean that a polynucleotide, polypeptide, orhydrocarbon is present in a different concentration in an engineeredhost cell as compared to its concentration in a corresponding wild-typecell under the same conditions. As used herein, the term “express” withrespect to a polynucleotide is to cause it to function. A polynucleotidewhich encodes a polypeptide (or protein) will, when expressed, betranscribed and translated to produce that polypeptide (or protein). Asused herein, the term “overexpress” means to express or cause to beexpressed a polynucleotide or polypeptide in a cell at a greaterconcentration than is normally expressed in a corresponding wild-typecell under the same conditions.

As used herein, the term “titer” refers to the quantity of a malonyl-CoAderived compound including a fatty acid derivative produced per unitvolume of host cell culture. In any aspect of the compositions andmethods described herein, a fatty acid derivative or other compound isproduced at a titer of about 25 mg/L, about 50 mg/L, about 75 mg/L,about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about200 mg/L, about 225 mg/L, about 250 mg/L, about 275 mg/L, about 300mg/L, about 325 mg/L, about 350 mg/L, about 375 mg/L, about 400 mg/L,about 425 mg/L, about 450 mg/L, about 475 mg/L, about 500 mg/L, about525 mg/L, about 550 mg/L, about 575 mg/L, about 600 mg/L, about 625mg/L, about 650 mg/L, about 675 mg/L, about 700 mg/L, about 725 mg/L,about 750 mg/L, about 775 mg/L, about 800 mg/L, about 825 mg/L, about850 mg/L, about 875 mg/L, about 900 mg/L, about 925 mg/L, about 950mg/L, about 975 mg/L, about 1000 mg/L, about 1050 mg/L, about 1075 mg/L,about 1100 mg/L, about 1125 mg/L, about 1150 mg/L, about 1175 mg/L,about 1200 mg/L, about 1225 mg/L, about 1250 mg/L, about 1275 mg/L,about 1300 mg/L, about 1325 mg/L, about 1350 mg/L, about 1375 mg/L,about 1400 mg/L, about 1425 mg/L, about 1450 mg/L, about 1475 mg/L,about 1500 mg/L, about 1525 mg/L, about 1550 mg/L, about 1575 mg/L,about 1600 mg/L, about 1625 mg/L, about 1650 mg/L, about 1675 mg/L,about 1700 mg/L, about 1725 mg/L, about 1750 mg/L, about 1775 mg/L,about 1800 mg/L, about 1825 mg/L, about 1850 mg/L, about 1875 mg/L,about 1900 mg/L, about 1925 mg/L, about 1950 mg/L, about 1975 mg/L,about 2000 mg/L (2 g/L), 3 g/L, 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L,50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or a range bounded byany two of the foregoing values. In other embodiments, a fatty acidderivative or other compound is produced at a titer of more than 100g/L, more than 200 g/L, or more than 300 g/L. One preferred titer offatty acid derivative or other compound produced by a recombinant hostcell according to the methods of the disclosure is from 5 g/L to 200g/L, 10 g/L to 150 g/L, 20 g/L to 120 g/L and 30 g/L to 100 g/L. Thetiter may refer to a particular fatty acid derivative or a combinationof fatty acid derivatives or another compound or a combination of othercompounds produced by a given recombinant host cell culture. Forexample, the expression of an ACC variant in a recombinant host cellsuch as E. coli results in the production of a higher titer as comparedto a recombinant host cell expressing the corresponding wild typepolypeptide. In one embodiment, the higher titer ranges from at leastabout 5 g/L to about 200 g/L.

As used herein, the “yield of a malonyl-CoA derived compound includingfatty acid derivatives or other compounds produced by a host cell”refers to the efficiency by which an input carbon source is converted toproduct (i.e., a malonyl-CoA derived compound including a fatty acidderivative and/or other compounds) in a host cell. Host cells engineeredto produce a malonyl-CoA derived compound including a fatty acidderivative according to the methods of the disclosure have a yield of atleast about 3%, at least about 4%, at least about 5%, at least about 6%,at least about 7%, at least about 8%, at least about 9%, at least about10%, at least about 11%, at least about 12%, at least about 13%, atleast about 14%, at least about 15%, at least about 16%, at least about17%, at least about 18%, at least about 19%, at least about 20%, atleast about 21%, at least about 22%, at least about 23%, at least about24%, at least about 25%, at least about 26%, at least about 27%, atleast about 28%, at least about 29%, or at least about 30% or a rangebounded by any two of the foregoing values. In other embodiments, afatty acid derivative or derivatives or other compound(s) are producedat a yield of more than about 30%, more than about 35%, more than about40%, more than about 45%, more than about 50%, more than about 55%, morethan about 60%, more than about 65%, more than about 70%, more thanabout 75%, more than about 80%, more than about 85%, more than about90%, more than 100%, more than 200%, more than 250%, more than 300%,more than 350%, more than 400%, more than 450%, more than 500%, morethan 550%, more than 600%, more than 650%, more than 700%, more than750%, or more. Alternatively, or in addition, the yield is about 30% orless, about 27% or less, about 25% or less, or about 22% or less. Inanother embodiment, the yield is about 50% or less, about 45% or less,or about 35% or less. In another embodiment, the yield is about 95% orless, or 90% or less, or 85% or less, or 80% or less, or 75% or less, or70% or less, or 65% or less, or 60% or less, or 55% or less, or 50% orless. Thus, the yield can be bounded by any two of the above endpoints.For example, the yield of a malonyl-CoA derived compound including afatty acid derivative or derivatives produced by the recombinant hostcell according to the methods of the disclosure can be about 5% to about15%, about 10% to about 25%, about 10% to about 22%, about 15% to about27%, about 18% to about 22%, about 20% to about 28%, about 20% to about30%, about 30% to about 40%, about 40% to about 50%, about 50% to about60%, about 60% to about 70%, about 70% to about 80%, about 80% to about90%, about 90% to about 100%, about 100% to about 200%, about 200% toabout 300%, about 300% to about 400%, about 400% to about 500%, about500% to about 600%, about 600% to about 700%, or about 700% to about800%. The yield may refer to a particular malonyl-CoA derived compoundincluding a fatty acid derivative or a combination of fatty acidderivatives or another compound or another combination of compoundsproduced by a given recombinant host cell culture. In one embodiment,the expression of a an ACC variant in a recombinant host cell such as E.coli results in the production of a higher yield of malonyl-CoA derivedcompounds including fatty acid derivatives such as, for example, fattyesters as compared to a host cell expressing the corresponding wild typepolypeptide. In one embodiment, the higher yield ranges from about 10%to about 800% of theoretical yield.

As used herein, the term “productivity” refers to the quantity of amalonyl-CoA derived compound including a fatty acid derivative orderivatives or another compound or compounds produced per unit volume ofhost cell culture per unit time. In any aspect of the compositions andmethods described herein, the productivity of a malonyl-CoA derivedcompound including a fatty acid derivative or derivatives or othercompound or compounds produced by a recombinant host cell is at least100 mg/L/hour, at least 200 mg/L/hour, at least 300 mg/L/hour, at least400 mg/L/hour, at least 500 mg/L/hour, at least 600 mg/L/hour, at least700 mg/L/hour, at least 800 mg/L/hour, at least 900 mg/L/hour, at least1000 mg/L/hour, at least 1100 mg/L/hour, at least 1200 mg/L/hour, atleast 1300 mg/L/hour, at least 1400 mg/L/hour, at least 1500 mg/L/hour,at least 1600 mg/L/hour, at least 1700 mg/L/hour, at least 1800mg/L/hour, at least 1900 mg/L/hour, at least 2000 mg/L/hour, at least2100 mg/L/hour, at least 2200 mg/L/hour, at least 2300 mg/L/hour, atleast 2400 mg/L/hour, 2500 mg/L/hour, or as high as 10 g/L/hour(dependent upon cell mass). For example, the productivity of amalonyl-CoA derived compound including a fatty acid derivative orderivatives or other compound(s) produced by a recombinant host cellaccording to the methods of the disclosure may be from 500 mg/L/hour to2500 mg/L/hour, or from 700 mg/L/hour to 2000 mg/L/hour. Theproductivity may refer to a particular a malonyl-CoA derived compoundincluding a fatty acid derivative or a combination of fatty acidderivatives or other compound(s) produced by a given host cell culture.For example, the expression of an ACC variant in a recombinant host cellsuch as E. coli results in the production of an increased productivityof malonyl-CoA derived compounds including fatty acid derivatives orother compounds as compared to a recombinant host cell expressing thecorresponding wild type polypeptide. In one embodiment, the higherproductivity ranges from about 0.3 g/L/h to about 3 g/L/h to about 10g/L/h to about 100 g/L/h to about a 1000 g/L/h.

As used herein, the term “total fatty species” and “total fatty acidproduct” and “fatty acid derivative” may be used interchangeably hereinwith reference to the amount of fatty acid derivatives that can beproduced by the host cell that expresses the ACC variant, as evaluatedby GC-FID. The same terms may be used to mean, for example, fattyesters, fatty alcohols, fatty aldehydes, fatty amines, and free fattyacids when referring to a fatty acid derivative analysis.

As used herein, the term “glucose utilization rate” means the amount ofglucose used by the culture per unit time, reported as grams/liter/hour(g/L/hr).

As used herein, the term “carbon source” refers to a substrate orcompound suitable to be used as a source of carbon for prokaryotic orsimple eukaryotic cell growth. Carbon sources can be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO₂).Exemplary carbon sources include, but are not limited to,monosaccharides, such as glucose, fructose, mannose, galactose, xylose,and arabinose; oligosaccharides, such as fructo-oligosaccharide andgalacto-oligosaccharide; polysaccharides such as starch, cellulose,pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose,and turanose; cellulosic material and variants such as hemicelluloses,methyl cellulose and sodium carboxymethyl cellulose; saturated orunsaturated fatty acids, succinate, lactate, and acetate; alcohols, suchas ethanol, methanol, and glycerol, or mixtures thereof. The carbonsource can also be a product of photosynthesis, such as glucose. Incertain embodiments, the carbon source is biomass. In other embodiments,the carbon source is glucose. In other embodiments the carbon source issucrose. In other embodiments the carbon source is glycerol. In otherembodiments, the carbon source is a simple carbon source. In otherembodiments, the carbon source is a renewable carbon source.

As used herein, the term “biomass” refers to any biological materialfrom which a carbon source is derived. In some embodiments, a biomass isprocessed into a carbon source, which is suitable for bioconversion. Inother embodiments, the biomass does not require further processing intoa carbon source. The carbon source can be converted into a compositioncomprising fatty esters. Fatty esters find utility in a number ofproducts including, but not limited to, surfactants, polymers, films,textiles, dyes, pharmaceuticals, fragrances and flavoring agents,lacquers, paints, varnishes, softening agents in resins and plastics,plasticizers, flame retardants, and additives in gasoline and oil.

An exemplary source of biomass is plant matter or vegetation, such ascorn, sugar cane, or switchgrass. Another exemplary source of biomass ismetabolic waste products, such as animal matter (e.g., cow manure).Further exemplary sources of biomass include algae and other marineplants. Biomass also includes waste products from industry, agriculture,forestry, and households, including, but not limited to, glycerol,fermentation waste, ensilage, straw, lumber, sewage, garbage, cellulosicurban waste, and food leftovers (e.g., soaps, oils and fatty acids). Theterm “biomass” also can refer to sources of carbon, such ascarbohydrates (e.g., monosaccharides, disaccharides, orpolysaccharides).

As used herein, the term “isolated,” with respect to products (such asfatty acid derivatives) refers to products that are separated fromcellular components, cell culture media, or chemical or syntheticprecursors. The fatty acid derivatives produced by the methods describedherein can be relatively immiscible in the fermentation broth, as wellas in the cytoplasm. Therefore, the fatty acid derivatives can collectin an organic phase either intracellularly or extracellularly.

As used herein, the terms “purify,” “purified,” or “purification” meanthe removal or isolation of a molecule from its environment by, forexample, isolation or separation. “Substantially purified” molecules areat least about 60% free (e.g., at least about 70% free, at least about75% free, at least about 85% free, at least about 90% free, at leastabout 95% free, at least about 97% free, at least about 99% free) fromother components with which they are associated. As used herein, theseterms also refer to the removal of contaminants from a sample. Forexample, the removal of contaminants can result in an increase in thepercentage of malonyl-CoA derived compounds including fatty acidderivatives or other compounds in a sample. For example, when amalonyl-CoA derived compound including a fatty acid derivative or othercompound is produced in a recombinant host cell, the malonyl-CoA derivedcompound including the fatty acid derivative or other compound can bepurified by the removal of host cell proteins. After purification, thepercentage of malonyl-CoA derived compounds including fatty acidderivatives or other compounds in the sample is increased. The terms“purify,” “purified,” and “purification” are relative terms which do notrequire absolute purity. Thus, for example, when a malonyl-CoA derivedcompound (including a fatty acid derivative or other compound) isproduced in recombinant host cells, a malonyl-CoA derived compound(including a purified fatty acid derivative or other compound) is amalonyl-CoA derived compound (including a fatty acid derivative or othercompound) that is substantially separated from other cellular components(e.g., nucleic acids, polypeptides, lipids, carbohydrates, or otherhydrocarbons).

As used herein, the term “attenuate” means to weaken, reduce, ordiminish. For example, a polypeptide can be attenuated by modifying thepolypeptide to reduce its activity (e.g., by modifying a nucleotidesequence that encodes the polypeptide).

Acetyl-CoA Carboxylase (ACC) Variants

Fatty acid synthase (FAS) denotes a group of polypeptides that catalyzethe initiation and elongation of acyl chains (Marrakchi et al. (2002)Biochemical Society 30:1050-1055). The acyl carrier protein (ACP) alongwith the enzymes in the FAS pathway control the length, degree ofsaturation and branching of the fatty acids produced. Enzymes that areincluded in the FAS pathway include, but are not limited to, ACC, FabD,FabH, FabG, FabA, FabZ, FabI, FabK, FabL, FabM, FabB, and FabF.Depending upon the desired product one or more of these genes can beoptionally attenuated or over-expressed in a recombinant host cell (see,e.g., U.S. Pat. Nos. 8,658,404; 8,597,922; 8,535,916; 8,530,221;8,372,610; 8,323,924; 8,313,934; 8,283,143; 8,268,599; 8,183,028;8,110,670; 8,110,093; and 8,097,439).

The ACC enzyme (E.C. 6.4.1.2.) catalyzes the first committed step offatty acid biosynthesis, the carboxylation of acetyl-CoA to malonyl-CoA.As such, it provides the malonyl-CoA substrate for the biosynthesis offatty acids, fatty acid derivatives and other non-fatty acid compounds(see, e.g., FIG. 4). The ACC enzyme is found in most living organismsand presents as a multi-subunit enzyme in the majority of allprokaryotes and in the chloroplasts of most plants and algae. Theprokaryotic ACC enzyme or ACC enzyme complex includes four differentproteins encoded by four different genes (i.e., accA, accB, accC, andaccD) that assemble into a complex at a fixed ratio (Broussard et al.(2013) supra). The genes accB and accC encode ACC subunits biotincarboxyl carrier protein (BCCP) and biotin carboxylase (BC),respectively. The present disclosure surprisingly shows that mutation(s)in only one or two of the ACC genes (e.g., accB, accC) is sufficient toincrease the fatty acid flux and result in a higher titer and/or yieldof malonyl-CoA derived compounds including fatty acid derivatives suchas fatty acid methyl esters (FAME). The present disclosure shows thatmutations in the coding region of the accB gene are beneficial, and thatsimultaneous expression changes of both accB and accC genes are alsobeneficial. In E. coli, the accB and accC genes are found adjacent in anoperon in the chromosome. The ACC variants disclosed herein containmutations or expression changes in one or two of the four ACC genes,which is sufficient to confer increased ACC enzymatic activity ontocells that already contain the other ACC subunit genes and polypeptides.

Thus, the present disclosure relates to, inter alia, ACC variants thatresult in a higher titer and/or higher yield of malonyl-CoA derivedcompounds when expressed in a cell; polypeptide sequences of such ACCvariants and functional fragments thereof; polynucleotides encoding ACCvariant polypeptide sequences; recombinant microorganisms includingnucleic acids encoding ACC variant polypeptides; microorganisms capableof expressing ACC variant polypeptides; cultures of such microorganisms;processes for producing malonyl-CoA-derived compounds including fattyacid derivatives and non-fatty acid compounds; and the resultantcompositions. Particularly, ACC variant polypeptides and microorganismsexpressing these polypeptides as well as related methods are providedherein. Examples of ACC variants are BCCP variants as shown in Tables 1and 3 (infra).

The E. coli wild-type nucleic acid sequence of accB is shown in SEQ IDNO: 1. The corresponding E. coli wild-type amino acid sequence for BCCP(encoded by accB of SEQ ID NO: 1) is shown in SEQ ID NO: 2. SEQ ID NO: 2was used as a template to generate the improved ACC variant polypeptidesin order to illustrate the disclosure (see Example 1, infra). Apreferred ACC variant has at least about 50%, about 60%, about 70%,about 80%, about 90%, or about 99% sequence identity to the amino acidsequence of the wild type E. coli of SEQ ID NO: 2. The first amino acidafter the ATG is designated amino acid “2”.

In one aspect, the disclosure provides ACC variant polypeptides or ACCvariants and nucleotide sequences that encode them. Various mutations indifferent amino acid positions (or residues) will increase production ofvarious fatty acid derivatives such as, for example, fatty acid methylester (FAME) production. For example, techniques such as targetedsite-saturation mutagenesis can be used to determine which positions andmutations provide the greatest improvement.

The wild type accB gene contains a GAT codon at position 2 encodingaspartic acid or aspartate (Asp, D). In one embodiment, it can be seenthat mutations in the amino acid position 2 within the wild-type accBgene of SEQ ID NO: 2 improved FAME production. These variant ACCpolypeptides (i.e., encoded by the mutant accB gene) are capable ofincreasing the production of fatty esters relative to wild-type ACC.Table 1 below depicts a summary of the best variants for accB position2. The skilled artisan will appreciate that increasing ester productionis one way to test the variant ACC polypeptides for their ability toincrease malonyl-CoA derived compounds such as, for example, fatty acidderivative production. Fatty ester production (rather than any otherfatty acid derivative production such as, for example, fatty alcoholproduction or fatty aldehyde production or fatty acid production, etc.)was used for illustrative purposes only and is not meant to limit thepresent disclosure. One of skill will recognize that other compoundsderived from malonyl-CoA can also be increased by following the teachingof the present disclosure and by employing the methods and protocols asdisclosed herein and generally available to those of skill in the art.

TABLE 1 accB Variants with Increased FAME Production Amino Acid MutantNucleic Acid Amino Acid Well Titer Codon Mutation Change Number SEQ IDNO: SEQ ID NO: C12 630% CAC D2H Histidine (H) 1 SEQ ID NO: 3 SEQ ID NO:4 C01 578% AAC D2N Asparagine (N) 2 SEQ ID NO: 5 SEQ ID NO: 6 F07 527%CAT D2H Histidine (H) 3 SEQ ID NO: 7 SEQ ID NO: 8 E05 475% ATT D2IIsoleucine (I) 4 SEQ ID NO: 9 SEQ ID NO: 10 F08 420% ATT D2I Isoleucine(I) 5 SEQ ID NO: 11 SEQ ID NO: 12 F06 331% ACT D2T Threonine (T) 6 SEQID NO: 13 SEQ ID NO: 14 B01 305% TCT D2S Serine (S) 7 SEQ ID NO: 15 SEQID NO: 16 E12 282% AGC D2S Serine (S) 8 SEQ ID NO: 17 SEQ ID NO: 18 C06276% CGA D2R Arginine (R) 9 SEQ ID NO: 19 SEQ ID NO: 20 F10 276% TCT D2SSerine (S) 10 SEQ ID NO: 21 SEQ ID NO: 22 B05 265% TAT D2Y Tyrosine (Y)11 SEQ ID NO: 23 SEQ ID NO: 24 B08 260% TCA D2S Serine (S) 12 SEQ ID NO:25 SEQ ID NO: 26 C04 255% TAC D2Y Tyrosine (Y) 13 SEQ ID NO: 27 SEQ IDNO: 28 G05 254% TAC D2Y Tyrosine (Y) 14 SEQ ID NO: 29 SEQ ID NO: 30 E08239% CTT D2L Leucine (L) 15 SEQ ID NO: 31 SEQ ID NO: 32 E02 234% CGA D2RArginine (R) 16 SEQ ID NO: 33 SEQ ID NO: 34 H12 225% TTG D2L Leucine (L)17 SEQ ID NO: 35 SEQ ID NO: 36 B03 223% CGA D2R Arginine (R) 18 SEQ IDNO: 37 SEQ ID NO: 38 A10 219% ACG D2T Threonine (T) 19 SEQ ID NO: 39 SEQID NO: 40 B02 217% TAT D2Y Tyrosine (Y) 20 SEQ ID NO: 41 SEQ ID NO: 42F03 214% CTT D2L Leucine (L) 21 SEQ ID NO: 43 SEQ ID NO: 44 G04 214% TTAD2L Leucine (L) 22 SEQ ID NO: 45 SEQ ID NO: 46 H10 214% CAG D2QGlutamine (Q) 23 SEQ ID NO: 47 SEQ ID NO: 48 F12 212% TAT D2Y Tyrosine(Y) 24 SEQ ID NO: 49 SEQ ID NO: 50 G03 206% TTA D2L Leucine (L) 25 SEQID NO: 51 SEQ ID NO: 52 B07 204% TTA D2L Leucine (L) 26 SEQ ID NO: 53SEQ ID NO: 54 B10 202% TTA D2L Leucine (L) 27 SEQ ID NO: 55 SEQ ID NO:56 C07 193% TTA D2L Leucine (L) 28 SEQ ID NO: 57 SEQ ID NO: 58 H05 177%TTG D2L Leucine (L) 29 SEQ ID NO: 59 SEQ ID NO: 60 F11 172% TTG D2LLeucine (L) 30 SEQ ID NO: 61 SEQ ID NO: 62 F04 168% CTT D2L Leucine (L)31 SEQ ID NO: 63 SEQ ID NO: 64 E06 146% ATC D2I Isoleucine (I) 32 SEQ IDNO: 65 SEQ ID NO: 66 E03 145% TAT D2Y Tyrosine (Y) 33 SEQ ID NO: 67 SEQID NO: 68 G07 144% GAA D2E Glutamate (E) 34 SEQ ID NO: 69 SEQ ID NO: 70A08 142% CTC D2L Leucine (L) 35 SEQ ID NO: 71 SEQ ID NO: 72 H07 130% CTCD2L Leucine (L) 36 SEQ ID NO: 73 SEQ ID NO: 74 A12 129% CTC D2L Leucine(L) 37 SEQ ID NO: 75 SEQ ID NO: 76 A01 126% ATC D2I Isoleucine (I) 38SEQ ID NO: 77 SEQ ID NO: 78 G06 126% GAA D2E Glutamate (E) 39 SEQ ID NO:79 SEQ ID NO: 80 H02 116% TCG D2S Serine (S) 40 SEQ ID NO: 81 SEQ ID NO:82 H06 114% TCG D2S Serine (S) 41 SEQ ID NO: 83 SEQ ID NO: 84 H08 114%TCG D2S Serine (S) 42 SEQ ID NO: 85 SEQ ID NO: 86 A11 107% TCG D2SSerine (S) 43 SEQ ID NO: 87 SEQ ID NO: 88 E01 106% TCG D2S Serine (S) 44SEQ ID NO: 89 SEQ ID NO: 90

Depending upon the position mutated, single or multiple amino acidchanges at specified positions give rise to increases in fatty acidderivative production as well as increases in the production ofnon-fatty acid compounds. In one embodiment, a single or multiple aminoacid change results in an increase in fatty acid production. In anotherembodiment, a single or multiple amino acid change results in anincrease in fatty ester production, including but not limited to, fattyacid methyl ester (FAME) and/or fatty acid ethyl ester (FAEE). Inanother embodiment, a single or multiple amino acid change results in anincrease in fatty aldehye production. In another embodiment, a single ormultiple amino acid change results in an increase in fatty alcoholproduction. In another embodiment, a single or multiple amino acidchange results in an increase in fatty amine production. In anotherembodiment, a single or multiple amino acid change results in anincrease in hydrocarbon production. In another embodiment, a single ormultiple amino acid change results in an increase in alkane production.In another embodiment, a single or multiple amino acid change results inan increase in alkene or olefin production. In still another embodiment,a single or multiple amino acid change results in an increase inbifunctional fatty acid production, including but not limited to,hydroxy fatty acids and/or diacids. In yet another embodiment, a singleor multiple amino acid change results in an increase in bifunctionalfatty alcohol production. In still another embodiment, a single ormultiple amino acid change results in an increase in bifunctional fattyester and/or fatty amine production. In another embodiment, a single ormultiple amino acid change results in an increase in the production ofbeta-hydroxy fatty acid derived compounds. In another embodiment, asingle or multiple amino acid change results in an increase in theproduction of unsaturated fatty acid derived compounds. In still anotherembodiment, a single or multiple amino acid change results in anincrease in the production of flavanones and/or flavonoids. In anotherembodiment, a single or multiple amino acid change results in anincrease in the production of polyketides. In another embodiment, asingle or multiple amino acid change results in an increase in theproduction of 3-hydroxypropionic acid (3-HP). In another embodiment, asingle or multiple amino acid change results in an increase in theproduction of malonic acid or malonate.

Thus, combinations of one or more amino acid changes at specifiedpositions may give rise to increases in fatty acid derivative and/orfree fatty acid production and/or non-fatty acid based compounds such asflavanones and/or flavonoids, polyketides, malonate, 3-hydroxypropionicacid (3-HP), and others. The effect of each individual amino acid changeon fatty acid derivative production may or may not be additive to theeffect of other individual amino acid changes on fatty acid derivativeproduction or the production of non-fatty acid compounds. In someembodiments, a combination of one or more amino acid changes atspecified positions results in an increase in fatty acid derivativeproduction. Accordingly, one or multiple amino acid changes at specifiedpositions can give rise to increases in fatty acid derivativeproduction. Similarly, one or multiple amino acid changes at specifiedpositions can give rise to increases in non-fatty acid compounds.

In addition to the ACC variants show in Table 1 above, an error pronelibrary of the accB gene was built and screened using SEQ ID NO: 1 as atemplate. Additional accB variants were identified by introducing singleor multiple mutations (see Example 1, Table 3, infra). Thus, 63beneficial mutations (see Tables 1 and 3) were identified in the codingregion of accB that resulted in an increased titer of FAME. Notably, ahigh number of mutations were found in the N-terminal amino acid regionthat ranges from about amino acid position 1 to about position 60.

In one aspect, the disclosure relates to ACC variant polypeptides withat least about 50% sequence identity to SEQ ID NO: 2. In someembodiments, a variant ACC polypeptide shows at least about 50%, (e.g.,about 48% to about 52%), at least about 60%, at least about 70%, atleast about 75%, at least 76%, at least about 77%, at least about 78%,at least about 79%, at least about 80%, at least about 81%, at leastabout 82%, at least about 83%, at least about 84%, at least about 85%,at least about 86%, at least about 87%, at least about 88%, at leastabout 89%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least 99%sequence identity to the wild-type ACC sequence of SEQ ID NO: 2 and alsoincludes one or more substitutions which result in usefulcharacteristics and/or properties as described herein. In one aspect ofthe disclosure, the ACC variant polypeptide with improvedcharacteristics has about 100% sequence identity to SEQ ID NO: 6. Inanother aspect of the disclosure, the ACC variant polypeptide has about100% sequence identity to any one of the following SEQ ID NOS,including, but not limited to, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ IDNO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:86, SEQ ID NO: 88, and SEQ ID NO: 90.

In a related aspect, an ACC variant polypeptide is encoded by anucleotide sequence having 100% sequence identity to SEQ ID NO: 5. Inanother related aspect, an ACC variant polypeptide is encoded by anucleotide sequence having about 100% sequence identity to any one ofthe following SEQ ID NOS including, but not limited to, SEQ ID NO: 3,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ IDNO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89.

In another aspect, the disclosure relates to ACC variant polypeptideswith improved ACC activity with at least about 50% sequence identity toSEQ ID NO: 6. In some embodiments, an ACC variant polypeptide has atleast about 50%, (e.g., about 48% to about 52%), at least about 60%, atleast about 70%, at least about 75%, at least 76%, at least about 77%,at least about 78%, at least about 79%, at least about 80%, at leastabout 81%, at least about 82%, at least about 83%, at least about 84%,at least about 85%, at least about 86%, at least about 87%, at leastabout 88%, at least about 89%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least 99% sequence identity to the ACC variant sequence of SEQ IDNO: 6 and also includes one or more substitutions which results inimproved characteristics and/or properties as described herein. Inanother aspect, the disclosure relates to ACC variant polypeptides withat least about 50% sequence identity to any one of the following SEQ IDNOS including, but not limited to, SEQ ID NO: 4, SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ IDNO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO:76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ IDNO: 86, SEQ ID NO: 88, and SEQ ID NO: 90. In some embodiments, an ACCvariant polypeptide has at least about 50%, (e.g., about 48% to about52%), at least about 60%, at least about 70%, at least about 75%, atleast 76%, at least about 77%, at least about 78%, at least about 79%,at least about 80%, at least about 81%, at least about 82%, at leastabout 83%, at least about 84%, at least about 85%, at least about 86%,at least about 87%, at least about 88%, at least about 89%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least 99% sequence identity to theACC sequence of any one of the following SEQ ID NOS including, but notlimited to, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ IDNO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ IDNO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQID NO: 88, and SEQ ID NO: 90, which encompass one or more substitutionsthat result in improved characteristics and/or properties as describedherein.

In another aspect, the disclosure relates to ACC variant polypeptidesthat include an amino acid sequence encoded by a nucleic acid sequencethat has at least about 70%, at least about 75%, at least 76%, at leastabout 77%, at least about 78%, at least about 79%, at least about 80%,at least about 81%, at least about 82%, at least about 83%, at leastabout 84%, at least about 85%, at least about 86%, at least about 87%,at least about 88%, at least about 89%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least 99% sequence identity to the ACC variant sequenceof SEQ ID NO: 5. In some embodiments the nucleic acid sequence encodesan ACC variant with one or more substitutions which results in improvedcharacteristics and/or properties as described herein. In otherembodiments, the variant ACC nucleic acid sequence is derived from anorganism such as E. coli. In another aspect, the disclosure relates toACC variant polypeptides that include an amino acid sequence encoded bya nucleic acid sequence that has at least about 70%, at least about 75%,at least 76%, at least about 77%, at least about 78%, at least about79%, at least about 80%, at least about 81%, at least about 82%, atleast about 83%, at least about 84%, at least about 85%, at least about86%, at least about 87%, at least about 88%, at least about 89%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least 99% sequence identityto the ACC variant sequence of any one of the following SEQ ID NOSincluding, but not limited to, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ IDNO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,SEQ ID NO: 87, and SEQ ID NO: 89. In some embodiments the nucleic acidsequence encodes an ACC variant with one or more substitutions whichresults in improved characteristics and/or properties as describedherein. In other embodiments, the ACC variant nucleic acid sequence isderived from an organism such as E. coli.

In another aspect, the disclosure relates to ACC variant polypeptidesthat include an amino acid sequence encoded by a nucleic acid thathybridizes under stringent conditions over substantially the entirelength of a nucleic acid corresponding to any one of the following SEQID NOS including, but not limited to, SEQ ID NO:3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ IDNO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO:83, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89. In some embodimentsthe nucleic acid sequence encodes an ACC variant nucleic acid sequencederived from an organism such as E. coli. In a related aspect, thedisclosure provides ACC variants encoded by a nucleotide sequence havingat least about 70%, at least about 75%, at least 76%, at least about77%, at least about 78%, at least about 79%, at least about 80%, atleast about 81%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least 99% sequence identity to SEQ ID NO: 1 and comprises oneor more of the substitutions disclosed herein.

The present disclosure shows that mutations in the coding region of theaccB gene alone are beneficial (supra) while simultaneous expressionchanges of both accB and accC genes are also beneficial. In E. coli, theaccB and accC genes are found adjacent in an operon in the chromosome.Thus, an expression library of the accBC operon was built and screenedfor variants that showed improvement in the ACC activity (i.e., measuredby the increased production of malonyl-CoA derived compounds in a cell)over the wild type accBC promoter. Table 2 (supra) depicts a summary ofthe best variants based on an accBC T5 promoter sequence as shown below(underlined regions indicate −35 and −10 positions of the promoters):

E. coli wild-type accBC promoter (PaccBC) regionnucleotide sequence (SEQ ID NO: 91):TTGTTGCAAATTACACGGTGTTGAAGGTTATTTACATGTTAGCTGTTGATTATCTTCCCTGATAAGACCAGTATTTAGCTBacteriophage T5 promoter (PT5) nucleotide sequence (SEQ ID NO: 92):AATCATAAAAAATTTATTTGCTTTCAGGAAAATTTTTCTGTATAATAGAT TC

TABLE 2 accBC T5 Promoter Variants with Increased FAME Production WellTiter accBC T5 Promoter Sequence SEQ ID NO: G06 315%AATCATAAAAAATTTATTTGCTCTCAGGAAAATTTTTCTGGATAATAGATTC 93 E09 292%AATCATAAAAAATTTATCTTCTCTCAGGAAAATTTTTCTGTATTATAGATTC 94 F06 226%AATCATAAAAAATTTATCTGCCTTCAGGAAAATTTTTCTGTATAATAGATTC 95 B11 219%AATCATAAAAAATTTATTTGCCTTCAGGAAAATTTTTCTGTATAGTAGATTC 96

The library can be built using primers that replace the native accBCpromoter region with any suitable promoter library (e.g., hybridpromoter, artificial or synthetic promoter, promoter from differentorganism, promoter from different gene in same organism, commercialpromoter, etc.), which usually contains degenerate nucleotides tointroduce random mutations. In certain embodiments, the promoter is adevelopmentally-regulated, an organelle-specific, a tissue-specific, aninducible, a constitutive, or a cell-specific promoter. All suitablepromoters are contemplated herein. In other embodiments, expressionchanges to the accBC operon can be made using numerous techniques knownto those of skill in the art, including, but not limited to, replacementof the promoter with a different E. coli promoter or heterologouspromoter, mutation of the native promoter, mutations in the ribosomebinding sites (RBS) of accB and accC separately or together, alterationof the untranslated region (UTR) between the accBC promoter and the accBgene, duplication of the accBC operon in the chromosome or on a plasmid,replacement of the chromosomal accBC operon with a plasmid-encodedoperon, engineering of the transcription factors which bind the accBCpromoter region. In one exemplary embodiment, a bacteriophage T5promoter is used. In one embodiment, the promoter library can be joinedto appropriate homology regions using a PCR technique, and the librarycan then be integrated into the bacterial chromosome (see Example 2),replacing the native accBC promoter. The expression library can bescreened as shown in Example 2 (infra).

Improved Properties of ACC Variants

The wild type BCCP (SEQ ID NOS: 1 and 2) was genetically altered viamutagenesis to produce a high percentage of malonyl-CoA derivedcompounds such as FAME without the need to overexpress any other geneusing expression in E. coli as an illustrative model (see Example 1,infra). The same was accomplished by genetically altering the accBCoperon (see Example 2, infra). Thus, when expressed in a recombinanthost cell such as E. coli, variants of the wild type BCCP result in ahigher titer and yield of the desired product, i.e., they producegreater amounts of malonyl-CoA derived compounds such as fatty acidderivatives when expressed in a host cell (i.e., a recombinant cell)compared to the wild type host cell (that does not express the ACCvariant). The wild type ACC is a native protein complex and it normallyrequires all four proteins for its activity, including, biotincarboxylase (BC), biotin carboxyl carrier protein (BCCP), and twoproteins that form the carboxyltransferase (CT). However, the variantBCCPs of the present disclosure appear to confer onto the cell theability to increase the production of malonyl-CoA derived compounds.Without wishing to be bound by theory, it is contemplated that this maybe a direct consequence of the variant BCCPs directly or indirectlyconferring increased ACC activity in cells that express native ACC. Forexample, the BCCP variant polypeptides produced from about 100% to about650% FAME when expressed in host cells (see Table 1, supra, and Table 3,infra) compared to the wild type cells. This means that the observedtiter of FAME ranged from up to 650% of the FAME titer normally producedby the wild type cell. In another example, changes in the accBC operonlead to variant BCCP polypeptides that produced from about 200% to about350% FAME titer when expressed in host cells (see Table 2, supra)compared to wild type cells.

In one embodiment, the ACC variant polypeptides are expected to produceincreased amounts of fatty acid derivatives including, but not limitedto, fatty esters such as fatty acid methyl esters (FAME) and fatty acidethyl esters (FAEE), fatty amines, fatty aldehydes, fatty alcohols,short and long chain alcohols, hydrocarbons, ketones, alkanes, terminalolefins, internal olefins, beta hydroxy fatty acid derivatives,bifunctional fatty acid derivatives, and unsaturated fatty acidderivatives compared to the wild type ACC enzyme. In another embodiment,the ACC variant polypeptides are expected to produce increased amountsof non-fatty acid based compounds (e.g., flavanones and flavonoids,polyketides, 3-hydroxypropionic acid, malonate, etc.) compared to thewild type ACC enzyme. One of skill will recognize that the end productsthat can be produced through the ACC variants encompass several classesof compounds including fatty acid derivatives and non-fatty acidcompounds, depending on the various biochemical pathways that areinfluenced by the upregulation of malonyl-CoA. FIG. 4 providesnon-exhaustive examples of possible compounds.

Methods of Making ACC Variants

In practicing the methods of the present disclosure, mutagenesis is usedto prepare groups of recombinant host cells for screening. Typically,the recombinant host cells comprise one or more polynucleotide sequencesthat include an open reading frame for an ACC variant polypeptide, suchas a variant accB gene together with operably-linked regulatorysequences and/or an accB gene with operably-linked variant accBCpromoter(s). Numerous examples of variant ACC polypeptides includingvariant BCCP polypeptides useful in the practice of the methods of thepresent disclosure are described herein. Examples of regulatorysequences useful in the practice of the methods of the presentdisclosure are also described herein. Mutagenesis of such polynucleotidesequences can be performed using genetic engineering techniques, such assite directed mutagenesis, random chemical mutagenesis, exonuclease IIIdeletion procedures, or standard cloning techniques. Alternatively,mutations in polynucleotide sequences can be created using chemicalsynthesis or modification procedures. Those of ordinary skill in the artwill recognize that the protocols and procedures employed herein can bemodified and that such modifications are in accordance with thevariations of the disclosure. For example, when method steps aredescribed in a certain order, the ordering of steps can be modifiedand/or performed in parallel or sequentially.

Mutagenesis methods are well known in the art and include, for example,the following. In error prone PCR (Leung et al. (1989) Technique1:11-15; and Caldwell et al. (1992) PCR Methods Applic. 2:28-33), PCR isperformed under conditions where the copying fidelity of the DNApolymerase is low, such that a high rate of point mutations is obtainedalong the entire length of the PCR product. Briefly, in such procedures,polynucleotides to be mutagenized are mixed with PCR primers, reactionbuffer, MgCl₂, MnCl₂, Taq polymerase, and an appropriate concentrationof dNTPs for achieving a high rate of point mutation along the entirelength of the PCR product. For example, the reaction can be performedusing 20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCRprimer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3),and 0.01% gelatin, 7 mM MgCl₂, 0.5 mM MnCl₂, 5 units of Taq polymerase,0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performedfor 30 cycles of 94° C. for 1 min., 45° C. for 1 min., and 72° C. for 1min. It will be appreciated that these parameters can be varied asappropriate. The mutagenized polynucleotides are then cloned into anappropriate vector and the activities of the affected polypeptidesencoded by the mutagenized polynucleotides are evaluated. Mutagenesiscan also be performed using oligonucleotide directed mutagenesis(Reidhaar-Olson et al. (1988) Science 241:53-57) to generatesite-specific mutations in any cloned DNA of interest. Briefly, in suchprocedures a plurality of double stranded oligonucleotides bearing oneor more mutations to be introduced into the cloned DNA are synthesizedand assembled into the cloned DNA to be mutagenized. Clones containingthe mutagenized DNA are recovered, and the activities of affectedpolypeptides are assessed. Another mutagenesis method for generatingpolynucleotide sequence variants is assembly PCR. Assembly PCR involvesthe assembly of a PCR product from a mixture of small DNA fragments. Alarge number of different PCR reactions occur in parallel in the samevial, with the products of one reaction priming the products of anotherreaction. Assembly PCR is described in, for example, U.S. Pat. No.5,965,408. Still another mutagenesis method of generating polynucleotidesequence variants is sexual PCR mutagenesis (Stemmer (1994) PNAS, USA91:10747-10751). In sexual PCR mutagenesis, forced homologousrecombination occurs between DNA molecules of different, but highlyrelated, DNA sequence in vitro as a result of random fragmentation ofthe DNA molecule based on sequence homology. This is followed byfixation of the crossover by primer extension in a PCR reaction.

ACC variants can also be created by in vivo mutagenesis. In someembodiments, random mutations in a nucleic acid sequence are generatedby propagating the polynucleotide sequence in a bacterial strain, suchas an E. coli strain, which carries mutations in one or more of the DNArepair pathways. Such “mutator” strains have a higher random mutationrate than that of a wild-type strain. Propagating a DNA sequence in oneof these strains will eventually generate random mutations within theDNA. Mutator strains suitable for use for in vivo mutagenesis aredescribed in, for example, PCT International Publication No. WO91/16427.

ACC variants can also be generated using cassette mutagenesis. Incassette mutagenesis, a small region of a double stranded DNA moleculeis replaced with a synthetic oligonucleotide “cassette” that differsfrom the starting polynucleotide sequence. The oligonucleotide oftencontains completely and/or partially randomized versions of the startingpolynucleotide sequence. There are many applications of cassettemutagenesis; for example, preparing mutant proteins by cassettemutagenesis (Richards, J. H. (1986) Nature 323:187; Ecker et al. (1987)1 Biol. Chem. 262:3524-3527); codon cassette mutagenesis to insert orreplace individual codons (Kegler-Ebo et al. (1994) Nucleic Acids Res.22(9):1593-1599); preparing variant polynucleotide sequences byrandomization of non-coding polynucleotide sequences comprisingregulatory sequences (e.g., ribosome binding sites, see, e.g., Barricket al. (1994) Nucleic Acids Res. 22(7): 1287-1295); Wilson et al. (1994)Biotechniques 17:944-953).

Recursive ensemble mutagenesis (Arkin et al. (1992) PNAS, USA89:7811-7815) can also be used to generate polynucleotide sequencevariants. Recursive ensemble mutagenesis is an algorithm for proteinengineering (i.e., protein mutagenesis) developed to produce diversepopulations of phenotypically related mutants whose members differ inamino acid sequence. This method uses a feedback mechanism to controlsuccessive rounds of combinatorial cassette mutagenesis. Exponentialensemble mutagenesis (Delegrave et al. (1993) Biotech. Res.11:1548-1552) can also be used to generate polynucleotide sequencevariants of ACC. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Random and site-directed mutagenesis can also beused (Arnold (1993) Curr. Opin. Biotech. 4:450-455).

Further, standard methods of in vivo mutagenesis can be used. Forexample, host cells, comprising one or more polynucleotide sequencesthat include an open reading frame for an ACC polypeptide, as well asoperably-linked regulatory sequences, can be subject to mutagenesis viaexposure to radiation (e.g., UV light or X-rays) or exposure tochemicals (e.g., ethylating agents, alkylating agents, or nucleic acidanalogs). In some host cell types, for example, bacteria, yeast, andplants, transposable elements can also be used for in vivo mutagenesis.

The mutagenesis of one or more polynucleotide sequences that encode anACC related polypeptide generally results in expression of an ACCpolypeptide product that demonstrates a modified and improved biologicalfunction. For example, the mutagenesis of one or more polynucleotidesequences that include an accB generally results in expression of a BCCPpolypeptide product that demonstrates a modified and improved biologicalfunction such as enhanced ACC activity. When preparing a group ofrecombinant microorganisms by mutagenesis of one or more polynucleotidesequences including the open reading frame encoding a BCCP andoperably-linked regulatory sequences, the protein expressed from theresulting mutagenized polynucleotide sequences will show increased ACCbiological function, Thus, an improved yield of malonyl-CoA derivedcompounds such as fatty acid derivatives or other compounds, and/orimproved compositions thereof including a modified mixture of fatty acidderivatives or other compounds (in terms of chain length, saturation,and the like) is observed upon culture of the recombinant microorganismunder conditions effective to express the mutant accB polynucleotide.

Hot Spots

The disclosure is also based, at least in part, on the identification ofcertain structurally conserved “hot spots” among variant ACCpolypeptides including variant BCCP polypeptides. Hot spots are regionswhere a high number of mutations are observed that lead to a highertiter of fatty acid derivatives such as FAME or a higher titer ofnon-fatty acid compounds. Notably, such regions are seen in variant BCCPpolypeptides, i.e., hot spots are observed in the N-terminal amino acidregion ranging from amino acid position 1 to about amino acid position60 (e.g., showing the highest number of mutations).

Motifs

The disclosure is also based, at least in part, on the identification ofcertain structurally conserved motifs among variant ACC polypeptidesincluding variant BCCP polypeptides. Biotin protein ligase (EC6.3.4.15), also known as holocarboxylase synthetase, catalyzes thecovalent attachment of the biotin prosthetic group to a specific lysineof the BCCP subunit of ACC. BCCP-type proteins have a conserved motif atthe site of biotin attachment. The motif includes K (lysine), which isthe biotinylated lysine residue. BCCP polypeptides of various bacterialspecies have this conserved motif, suggesting that any mutations in thatregion could result in a decreased function. The consensus sequence forthe motif is shown below, where K is the biotinylated lysine:

(L/I/V)E(A/V)MK(M/L)

FIG. 2 shows an alignment of a section of BCCP amino acid sequences fromseven different bacterial species, including Escherichia coli (SEQ IDNO: 97 (partial); SEQ ID NO: 2 (full); Accession Number NP 417721),Lactobacillus brevis (SEQ ID NO: 98 (partial); SEQ ID NO: 104 (full);Accession Number WP_011667655), Stenotrophomonas maltophilia (SEQ ID NO:99 (partial); SEQ ID NO: 105 (full); Accession Number AIL09846),Pseudomonas putida (SEQ ID NO: 100 (partial); SEQ ID NO: 106 (full);Accession Number AE016246_3), Bacillus subtilis (SEQ ID NO: 101(partial); SEQ ID NO: 107 (full); Accession Number NP_390315),Corynebacterium glutamicum (SEQ ID NO: 102 (partial); SEQ ID NO: 108(full); Accession Number WP 011013826), and Saccharomyces cerevisiae(SEQ ID NO: 103 (partial); SEQ ID NO: 109 (full); Accession NumberAAA20073). The motif is conserved across all seven species (see boxedregion on FIG. 2) regardless of an overall amino acid sequence identitythat ranges from about 10% percent to about 66% percent. For example,BCCP from Lactobacillus brevis showed a 28% identity when compared toEscherichia coli. BCCP from Stenotrophomonas maltophilia showed a 55%identity when compared to Escherichia coli. BCCP from Pseudomonas putidashowed a 66% identity when compared to Escherichia coli. BCCP fromBacillus subtilis showed a 40% identity when compared to Escherichiacoli. BCCP from Corynebacterium glutamicum and Saccharomyces cerevisiaeshowed a 10% identity when compared to Escherichia coli. This confirmsthat even in divergent species the motif is conserved. However, in someinstances, BCCP polypeptides have a high amino acid sequence identityacross various species, ranging from about 85% identity to about 100%identity. For example, BCCP from Escherichia alberti is about 98%identical to Escherichia coli; BCCP from Shigella flexneri is about 93%identical to Escherichia coli; and Klebsiella pneumonia is about 85%identical to Escherichia coli.

Host Cells and Host Cell Cultures

It should be appreciated, in view of the present disclosure, that any ofthe embodiments contemplated herein may be practiced with any host cellor microorganism that can be genetically modified via the introductionof one or more nucleic acid sequences that code for one or more ACCvariants. As such, the recombinant microorganisms of the disclosurefunction as host cells and encompass one or more polynucleotidesequences that include an open reading frame encoding a variant ACCpolypeptide conferring improved/increased ACC activity and/orimproved/increased production of a malonyl-CoA derived compound,together with operably-linked regulatory sequences that facilitateexpression of the ACC polypeptide in the host cell. In one embodiment,the polypeptide conferring improved/increased ACC activity and/orimproved/increased production of a malonyl-CoA derived compound is avariant or mutant of BCCP. In another embodiment, the polypeptideconferring improved/increased ACC activity and/or improved/increasedproduction of a malonyl-CoA derived compound is an improved BCCP orother improved ACC polypeptide or combination thereof resulting fromexpression changes in the accBC operon. In a recombinant host cell ofthe disclosure, the open reading frame coding sequences and/or theregulatory sequences may be modified relative to the correspondingwild-type coding sequence of the BCCP polypeptide. A fatty acidderivative composition is produced by culturing a host cell thatexpresses an ACC variant (i.e., a recombinant host cell) in the presenceof a carbon source under conditions effective to express the variant ACCpolypeptide including the variant BCCP (see FIGS. 1 and 3). Expressionof mutant or variant ACC polypeptides results in production of fattyacid derivative compositions with increased yields of fatty acids, fattyesters, fatty alcohols, fatty amines, fatty aldehydes, bifunctionalfatty acid derivatives, diacids, hydrocarbons, ketones, alkanes, alkenesor olefins, and/or the like. In one embodiment, expression of mutant orvariant ACC polypeptides such as variant BCCP polypeptides results inthe increased yield of fatty ester compositions including FAME and/orFAEE. A non-fatty acid compound is produced by culturing a host cellthat expresses an ACC variant (i.e., a recombinant host cell) in thepresence of a carbon source under conditions effective to express thevariant ACC polypeptide including the variant BCCP (see FIG. 4).Expression of mutant or variant ACC polypeptides results in productionof non-fatty acid compounds with increased yields including polyketides,flavanones, flavonoids, 3-hydroxypropionic acid (3-HP), malonate, andothers (see FIG. 4).

The host cells or microorganisms of the disclosure include host strainsor host cells that are genetically engineered to contain geneticalterations in order to test the efficiency of specific mutations onenzymatic activities (i.e., recombinant cells or microorganisms).Various optional genetic manipulations and alterations can be usedinterchangeably from one host cell to another, depending on what nativeenzymatic pathways are present in the original host cell. In oneembodiment, a host strain can be used for testing the ACC variants. Ahost strain may encompasses a number of genetic alterations in order totest specific variables and culture environments, including but notlimited to, culture conditions including fermentation components, carbonsource (e.g., feedstock), temperature, pressure, reduced culturecontamination conditions, and oxygen levels.

In one embodiment, a host strain called BD64 is used. BD64 is based onE. coli strain MG1655 that encompasses an optional fadE and fhuAdeletion. Acyl-CoA dehydrogenase (FadE) is an enzyme that is importantfor metabolizing fatty acids. It catalyzes the second step in fatty acidutilization (beta-oxidation), which is the process of breaking longchains of fatty acids (acyl-CoAs) into acetyl-CoA molecules. Morespecifically, the second step of the β-oxidation cycle of fatty aciddegradation in bacteria is the oxidation of acyl-CoA to 2-enoyl-CoA,which is catalyzed by FadE. When E. coli lacks FadE, it cannot grow onfatty acids as a carbon source but it can grow on acetate. The inabilityto utilize fatty acids of any chain length is consistent with thereported phenotype of fadE strains, i.e., fadE mutant strains where FadEfunction is disrupted. The fadE gene can be optionally knocked out orattenuated to assure that acyl-CoAs, which are intermediates in thispathway, can accumulate in the cell such that all acyl-CoAs can beefficiently converted to fatty esters by ester synthase. However, fadEattenuation is optional when sugar is used as a carbon source sinceunder such condition expression of FadE is likely repressed and FadEtherefore may only be present in small amounts and not able toefficiently compete with ester synthase for acyl-CoA substrates. FadE isrepressed due to catabolite repression. E. coli and many other microbesprefer to consume sugar over fatty acids, so when both sources areavailable sugar is consumed first by repressing the fad regulon (see D.Clark, J Bacteriol. (1981) 148(2):521-6)). Moreover, the absence ofsugars induces FadE expression. Acyl-CoA intermediates could be lost tothe beta oxidation pathway since the proteins expressed by the fadregulon (including FadE) are up-regulated and will efficiently competefor acyl-CoAs. Thus, it can be beneficial to have the fadE gene knockedout or attenuated. Since many carbon sources are sugar based, it isoptional to attenuate FadE. The gene fhuA codes for the TonA protein,which is an energy-coupled transporter and receptor in the outermembrane of E. coli (V. Braun (2009) J Bacteriol. 191(11):3431-3436).Its deletion is optional. The fhuA deletion allows the cell to becomemore resistant to phage attack which can be beneficial in certainfermentation conditions. Thus, it may be desirable to delete fhuA in ahost cell that is likely subject to potential contamination duringfermentation runs.

The host strain BD64 (supra) also encompasses optional overexpression ofone or more of the following genes: fadR from Escherichia coli, fabAfrom Salmonella typhimurium (NP_460041), fabD from Salmonellatyphimurium (NP 460164), fabG from Salmonella typhimurium (NP_460165),fabH from Salmonella typhimurium (NP 460163), fabV from Vibrio cholera(YP_001217283), and fabF from Clostridium acetobutylicum (NP 350156).The overexpression of one or more of these genes, which code for enzymesand regulators in fatty acid biosynthesis, can serve to further increasethe titer of fatty-acid derivative compounds under various cultureconditions.

In another embodiment, the wild-type E. coli strains MG1655 or W3110 areused as exemplary host cells for the production of fatty acidderivatives. Similarly, these host cells provide optional overexpressionof one or more biosynthesis genes (i.e., genes coding for enzymes andregulators of fatty acid biosynthesis) that can increase the titer offatty-acid derivative compounds under various culture conditions.Genetic alterations include fadR from Escherichia coli, fabA fromSalmonella typhimurium (NP_460041), fabD from Salmonella typhimurium(NP_460164), fabG from Salmonella typhimurium (NP_460165), fabH fromSalmonella typhimurium (NP_460163), fabV from Vibrio cholera(YP_001217283), and fabF from Clostridium acetobutylicum (NP_350156).

In some embodiments, the host cells or microorganisms that are used toexpress the variant ACC polypeptides will further express genes thatencompass certain enzymatic activities that can increase the productionto one or more particular fatty acid derivative(s) such as fatty esters,fatty alcohols, fatty amines, fatty aldehydes, bifunctional fatty acidderivatives, diacids, and the like (see FIGS. 1 and 3) as well asalkanes, alkenes or olefins, and ketones. In one embodiment, the hostcell has thioesterase activity (E.C. 3.1.2.* or E.C. 3.1.2.14 or E.C.3.1.1.5) for the production of fatty acids which can be increased byoverexpressing the gene. In another embodiment, the host cell has estersynthase activity (E.C. 2.3.1.75) for the production of fatty esters. Inanother embodiment, the host cell has acyl-ACP reductase (AAR) (E.C.1.2.1.80) activity and/or alcohol dehydrogenase activity (E.C. 1.1.1.1.)and/or fatty alcohol acyl-CoA reductase (FAR) (E.C. 1.1.1.*) activityand/or carboxylic acid reductase (CAR) (EC 1.2.99.6) activity for theproduction of fatty alcohols. In another embodiment, the host cell hasacyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity for the production offatty aldehydes. In another embodiment, the host cell has acyl-ACPreductase (AAR) (E.C. 1.2.1.80) activity and decarbonylase activity forthe production of alkanes and alkenes. In another embodiment, the hostcell has acyl-CoA reductase (E.C. 1.2.1.50) activity, acyl-CoA synthase(FadD) (E.C. 2.3.1.86) activity, and thioesterase (E.C. 3.1.2.* or E.C.3.1.2.14 or E.C. 3.1.1.5) activity for the production of fatty alcohols.In another embodiment, the host cell has ester synthase activity (E.C.2.3.1.75), acyl-CoA synthase (FadD) (E.C. 2.3.1.86) activity, andthioesterase (E.C. 3.1.2.* or E.C. 3.1.2.14 or E.C. 3.1.1.5) activityfor the production of fatty esters. In another embodiment, the host cellhas OleA activity for the production of ketones. In another embodiment,the host cell has OleBCD activity for the production of internalolefins. In another embodiment, the host cell has acyl-ACP reductase(AAR) (E.C. 1.2.1.80) activity and alcohol dehydrogenase activity (E.C.1.1.1.1.) for the production of fatty alcohols. In another embodiment,the host cell has thioesterase (E.C. 3.1.2.* or E.C. 3.1.2.14 or E.C.3.1.1.5) activity and decarboxylase activity for making terminalolefins. The expression of enzymatic activities in microorganisms andmicrobial cells is taught by U.S. Pat. Nos. 8,097,439; 8,110,093;8,110,670; 8,183,028; 8,268,599; 8,283,143; 8,232,924; 8,372,610; and8,530,221, which are incorporated herein by reference.

In other embodiments, the host cells or microorganisms that are used toexpress the variant ACC polypeptides will include certain native enzymeactivities that are upregulated or overexpressed in order to produce oneor more particular fatty acid derivative(s) such as fatty esters, fattyalcohols, fatty amines, fatty aldehydes, bifunctional fatty acidderivatives, diacids and the like (see FIG. 1). In one embodiment, thehost cell has a native thioesterase (E.C. 3.1.2.* or E.C. 3.1.2.14 orE.C. 3.1.1.5) activity for the production of fatty acids which can beincreased by overexpressing the thioesterase gene.

The present disclosure includes host strains or microorganisms thatexpress variant ACC polypeptide sequences including variant BCCPpolypeptide sequences. Examples of variant BCCP polypeptide sequencesthat when expressed in a host cell result in a higher titer of fattyacid derivatives including fatty esters include but are not limited to,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ IDNO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88,and SEQ ID NO: 90.

The recombinant host cell may produce a fatty ester, such as a fattyacid methyl ester (FAME) or a fatty acid ethyl ester (FAEE), a fattyalcohol, a fatty amine, a fatty aldehyde, a bifunctional fatty acidderivative, a diacids, an alkane, an olefin, a hydrocarbon, or the like;or a non-fatty acid compound such as a flavanone, a flavonoid, apolyketide, malonate, or 3-hydroxypropioic acid. The fatty acidderivatives or other compounds are typically recovered from the culturemedium and/or are isolated from the host cells. In one embodiment, thefatty acid derivatives or other compounds are recovered from the culturemedium (extracellular). In another embodiment, the fatty acidderivatives or other compounds are isolated from the host cells(intracellular). In another embodiment, the fatty acid derivatives orother compounds are recovered from the culture medium and isolated fromthe host cells. The fatty acid derivative composition produced by a hostcell can be analyzed using methods known in the art, for example,GC-FID, in order to determine the distribution of particular fatty acidderivatives as well as chain lengths and degree of saturation of thecomponents of the fatty acid derivative composition. Similarly, othercompounds can be analyzed through methods well known in the art.

Examples of host cells that function as microorganisms, include but arenot limited to cells from the genus Escherichia, Bacillus,Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus,Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces,Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus,Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas,Schizosaccharomyces, Yarrowia, or Streptomyces. In some embodiments, thehost cell is a Gram-positive bacterial cell. In other embodiments, thehost cell is a Gram-negative bacterial cell. In some embodiments, thehost cell is an E. coli cell. In other embodiments, the host cell is aBacillus lentus cell, a Bacillus brevis cell, a Bacillusstearothermophilus cell, a Bacillus lichenoformis cell, a Bacillusalkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell,a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillusclausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or aBacillus amyloliquefaciens cell.

In still other embodiments, the host cell is a Trichoderma koningiicell, a Trichoderma viride cell, a Trichoderma reesei cell, aTrichoderma longibrachiatum cell, an Aspergillus awamori cell, anAspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillusnidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, aHumicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacuscell, a Rhizomucor miehei cell, or a Mucor michei cell. In yet otherembodiments, the host cell is a Streptomyces lividans cell or aStreptomyces murinus cell. In yet other embodiments, the host cell is anActinomycetes cell. In some embodiments, the host cell is aSaccharomyces cerevisiae cell.

In other embodiments, the host cell is a cell from a eukaryotic plant,algae, cyanobacterium, green-sulfur bacterium, green non-sulfurbacterium, purple sulfur bacterium, purple non-sulfur bacterium,extremophile, yeast, fungus, an engineered organism thereof, or asynthetic organism. In some embodiments, the host cell islight-dependent or fixes carbon. In some embodiments, the host cell hasautotrophic activity.

In some embodiments, the host cell has photoautotrophic activity, suchas in the presence of light. In some embodiments, the host cell isheterotrophic or mixotrophic in the absence of light. In certainembodiments, the host cell is a cell from Arabidopsis thaliana, Panicumvirgatum, Miscanthus giganteus, Zea mays, Botryococcuse braunii,Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002,Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803,Thermosynechococcus elongates BP-1, Chlorobium tepidum, Chlorojlexusauranticus, Chromatiumm vinosum, Rhodospirillum rubrum, Rhodobactercapsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii,Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonasfluorescens, or Zymomonas mobilis.

In one embodiment, the microbial cell is from a cyanobacteria including,but not limited to, Prochlorococcus, Synechococcus, Synechocystis,Cyanothece, and Nostoc punctiforme. In another embodiment, the microbialcell is from a specific cyanobacterial species including, but notlimited to, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803,and Synechococcus sp. PCC7001.

Methods of Making Recombinant Host Cells and Cultures

Various methods well known in the art can be used to engineer host cellsto produce fatty acid derivatives and/or fatty acid derivativecompositions or other compounds. The methods can include the use ofvectors, preferably expression vectors, comprising a nucleic acidencoding a mutant or variant ACC including a mutant or variant BCCP, asdescribed herein. Those skilled in the art will appreciate a variety ofviral and non-viral vectors can be used in the methods described herein.

In some embodiments of the present disclosure, a higher titer of acompound such as a fatty acid ester in a particular composition is ahigher titer of a particular type of fatty acid ester or a combinationof fatty acid esters produced by a recombinant host cell culturerelative to the titer of the same fatty acid ester or combination offatty acid esters produced by a control culture of a correspondingwild-type host cell. In other embodiments, other fatty acid derivativesor non-fatty acid compounds are produced by the recombinant host cellculture in a similar fashion. In some embodiments, a mutant or variantACC polynucleotide (or gene) sequence including a mutant or variant accBpolynucleotide (or gene) sequence is provided to the host cell by way ofa recombinant vector, which comprises a promoter operably linked to thepolynucleotide sequence. In certain embodiments, the promoter is adevelopmentally-regulated, an organelle-specific, a tissue-specific, aninducible, a constitutive, or a cell-specific promoter. The recombinantvector typically comprises at least one sequence selected from anexpression control sequence operatively coupled to the polynucleotidesequence; a selection marker operatively coupled to the polynucleotidesequence; a marker sequence operatively coupled to the polynucleotidesequence; a purification moiety operatively coupled to thepolynucleotide sequence; a secretion sequence operatively coupled to thepolynucleotide sequence; and a targeting sequence operatively coupled tothe polynucleotide sequence. The polynucleotide sequences, comprisingopen reading frames encoding proteins and operably-linked regulatorysequences can be integrated into a chromosome of the recombinant hostcells, incorporated in one or more plasmid expression system resident inthe recombinant host cells, or both.

The expression vectors described herein include a polynucleotidesequence described herein in a form suitable for expression of thepolynucleotide sequence in a host cell. It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of polypeptide desired, etc. The expression vectorsdescribed herein can be introduced into host cells to producepolypeptides, including fusion polypeptides, encoded by thepolynucleotide sequences as described herein. Expression of genesencoding polypeptides in prokaryotes, for example, E. coli, is mostoften carried out with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionpolypeptides. Suitable expression systems for both prokaryotic andeukaryotic cells are well known in the art; see, e.g., Sambrook et al.,“Molecular Cloning: A Laboratory Manual,” second edition, Cold SpringHarbor Laboratory, (1989). In certain embodiments, a polynucleotidesequence of the disclosure is operably linked to a promoter derived frombacteriophage T5. In one embodiment, the host cell is a yeast cell. Inthis embodiment, the expression vector is a yeast expression vector.Vectors can be introduced into prokaryotic or eukaryotic cells via avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell. Suitable methods for transforming ortransfecting host cells can be found in, for example, Sambrook et al.(supra).

For stable transformation of bacterial cells, it is known that,depending upon the expression vector and transformation technique used,only a small fraction of cells will take-up and replicate the expressionvector. In order to identify and select these transformants, a gene thatencodes a selectable marker (e.g., resistance to an antibiotic) can beintroduced into the host cells along with the gene of interest.Selectable markers include those that confer resistance to drugs suchas, but not limited to, ampicillin, kanamycin, chloramphenicol, ortetracycline. Nucleic acids encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding apolypeptide described herein or can be introduced on a separate vector.Cells stably transformed with the introduced nucleic acid can beidentified by growth in the presence of an appropriate selection drug.

Culture and Fermentation of Recombinant Host Cells

As used herein, the term “fermentation” broadly refers to the conversionof organic materials into target substances by host cells, for example,the conversion of a carbon source by recombinant host cells into fattyacids or derivatives thereof by propagating a culture of the recombinanthost cells in a media comprising the carbon source. As used herein, theterm “conditions permissive for the production” means any conditionsthat allow a host cell to produce a desired product, such as amalonyl-CoA derived compound including fatty acid derivatives and othernon-fatty acid compounds. Similarly, the term “conditions in which thepolynucleotide sequence of a vector is expressed” means any conditionsthat allow a host cell to synthesize a polypeptide. Suitable conditionsinclude, for example, fermentation conditions. Fermentation conditionscan include many parameters, including but not limited to temperatureranges, levels of aeration, feed rates and media composition. Each ofthese conditions, individually and in combination, allows the host cellto grow. Fermentation can be aerobic, anaerobic, or variations thereof(such as micro-aerobic). Exemplary culture media include broths or gels.Generally, the medium includes a carbon source that can be metabolizedby a host cell directly. In addition, enzymes can be used in the mediumto facilitate the mobilization (e.g., the depolymerization of starch orcellulose to fermentable sugars) and subsequent metabolism of the carbonsource.

For small scale production, the engineered host cells can be grown inbatches of, for example, about 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 1mL, 5 mL, 10 mL, 15 mL, 25 mL, 50 mL, 75 mL, 100 mL, 500 mL, 1 L, 2 L, 5L, or 10 L; fermented; and induced to express a desired polynucleotidesequence, such as a polynucleotide sequence encoding an ACC variantpolypeptide. For large scale production, the engineered host cells canbe grown in cultures having a volume batches of about 10 L, 100 L, 1000L, 10,000 L, 100,000 L, 1,000,000 L or larger; fermented; and induced toexpress a desired polynucleotide sequence. The fatty acid derivativecompositions or other compounds described herein may be found in theextracellular environment of the recombinant host cell culture and canbe readily isolated from the culture medium. A fatty acid derivative maybe secreted by the recombinant host cell, transported into theextracellular environment or passively transferred into theextracellular environment of the recombinant host cell culture. In oneembodiment, a fatty ester composition may be isolated from a recombinanthost cell culture using routine methods known in the art. Any non-fattyacid compounds may be produced extracellularly or intracellularly.

Screening Recombinant Host Cells

In one embodiment of the present disclosure, the activity of a mutant orvariant ACC polypeptide is determined by culturing recombinant hostcells (comprising one or more mutagenized or variant ACC polynucleotidesequences), followed by screening to identify characteristics of, forexample, fatty acid derivative compositions or other compounds producedby the recombinant host cells; for example, titer, yield andproductivity of fatty acid derivatives or other compounds. In anotherembodiment, the activity of a mutant or variant ACC polypeptide isdetermined by culturing recombinant host cells (comprising one or moremutagenized or variant ACC polynucleotide sequences), followed byscreening to identify characteristics of, for example, fatty acidderivate compositions (e.g., fatty esters, fatty alcohols, fattyaldehydes, etc.) or other compounds produced by the recombinant hostcells; for example: titer, yield and productivity of fatty acidderivatives or other compounds. Mutant or variant ACC polypeptides ormutant or variant BCCP polypeptides and fragments thereof can be assayedfor improved ACC activity and/or improved/increased production of amalonyl-CoA derived compound using routine methods. For example, amutant or variant ACC polypeptide or BCCP polypeptide or fragmentthereof is contacted with a substrate (e.g., an acyl-CoA, an acyl-ACP, afree fatty acid, an alcohol) under conditions that allow the polypeptideto function. In one embodiment, a decrease in the level of the substrateor an increase in the level of a fatty ester or a fatty estercomposition can be measured to determine the ACC activity. The sameapplies to the production of fatty alcohols, fatty aldehydes, fattyamines and other fatty acid derivatives as well as other compounds.

Products Derived from Recombinant Host Cells

As used herein, “fraction of modem carbon” or fM has the same meaning asdefined by National Institute of Standards and Technology (NIST)Standard Reference Materials (SRMs 4990B and 4990C, known as oxalicacids standards HOxI and HOxII, respectively. The fundamental definitionrelates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD1950). This is roughly equivalent to decay-corrected pre-IndustrialRevolution wood. For the current living biosphere (plant material), fMis approximately 1.1.

Bioproducts (e.g., the fatty acid derivative compositions or non-fattyacid compositions produced in accordance with the present disclosure)including biologically produced organic compounds, and in particular,the fatty ester compositions produced using the fatty acid biosyntheticpathways described herein, have been produced from renewable carbonsources and, as such, are new compositions of matter. These newbioproducts can be distinguished from organic compounds derived frompetrochemical carbon on the basis of dual carbon-isotopic fingerprintingor ¹⁴C dating. Additionally, the specific source of biosourced carbon(e.g., glucose vs. glycerol) can be determined by dual carbon-isotopicfingerprinting (see, e.g., U.S. Pat. No. 7,169,588). The ability todistinguish bioproducts from petroleum based organic compounds isbeneficial in tracking these materials in commerce. For example, organiccompounds or chemicals comprising both biologically based and petroleumbased carbon isotope profiles may be distinguished from organiccompounds and chemicals made only of petroleum based materials. Hence,the bioproducts herein can be followed or tracked in commerce on thebasis of their unique carbon isotope profile. Bioproducts can bedistinguished from petroleum based organic compounds by comparing thestable carbon isotope ratio (¹³C/¹²C) in each sample. The ¹³C/¹²C ratioin a given bioproduct is a consequence of the ¹³C/¹²C ratio inatmospheric carbon dioxide at the time the carbon dioxide is fixed. Italso reflects the precise metabolic pathway. Regional variations alsooccur. Petroleum, C3 plants (the broadleaf), C4 plants (the grasses),and marine carbonates all show significant differences in ¹³C/¹²C andthe corresponding δ¹³C values. Both C4 and C3 plants exhibit a range of¹³C/¹²C isotopic ratios, but typical values are about −7 to about −13per mil for C4 plants and about −19 to about −27 per mil for C3 plants(see, e.g., Stuiver et al., Radiocarbon 19:355 (1977)). Coal andpetroleum fall generally in this latter range.

δ¹3C(‰)=[(¹³C/¹²C)sample−(¹³C/¹²C)standard]/(¹³C/¹²C)standard×1000

A series of alternative RMs have been developed in cooperation with theIAEA, USGS, NIST, and other selected international isotope laboratories.Notations for the per mil deviations from PDB is δ¹³C. Measurements aremade on CO₂ by high precision stable ratio mass spectrometry (IRMS) onmolecular ions of masses 44, 45, and 46. The compositions describedherein include fatty ester compositions and products produced by any ofthe methods described herein. Specifically, fatty ester composition orproduct can have a δ¹³C of about −28 or greater, about −27 or greater,−20 or greater, −18 or greater, −15 or greater, −13 or greater, −10 orgreater, or −8 or greater. For example, the fatty ester composition orproduct can have a δ¹³C of about −30 to about −15, about −27 to about−19, about −25 to about −21, about −15 to about −5, about −13 to about−7, or about −13 to about −10. In other instances, the fatty estercomposition or product t can have a δ¹³C of about −10, −11, −12, or−12.3. Fatty ester compositions and products produced in accordance withthe disclosure herein can also be distinguished from petroleum basedorganic compounds by comparing the amount of ¹⁴C in each compound.Because ¹⁴C has a nuclear half-life of 5730 years, petroleum based fuelscontaining “older” carbon can be distinguished from fatty estercompositions and bioproducts which contain “newer” carbon (see, e.g.,Currie, “Source Apportionment of Atmospheric Particles”,Characterization of Environmental Particles, J. Buffle and H. P. vanLeeuwen, Eds., 1 of Vol. I of the IUPAC Environmental AnalyticalChemistry Series (Lewis Publishers, Inc.) 3-74, (1992)).

The basic assumption in radiocarbon dating is that the constancy of ¹⁴Cconcentration in the atmosphere leads to the constancy of ¹⁴C in livingorganisms. However, because of atmospheric nuclear testing since 1950and the burning of fossil fuel since 1850, ¹⁴C has acquired a second,geochemical time characteristic. Its concentration in atmospheric CO₂,and hence in the living biosphere, approximately doubled at the peak ofnuclear testing, in the mid-1960s. It has since been gradually returningto the steady-state cosmogenic (atmospheric) baseline isotope rate(¹⁴C/¹²C) of about 1.2×10-12, with an approximate relaxation “half-life”of 7-10 years. (This latter half-life must not be taken literally;rather, one must use the detailed atmospheric nuclear input/decayfunction to trace the variation of atmospheric and biospheric¹⁴C sincethe onset of the nuclear age.) It is this latter biospheric¹⁴C timecharacteristic that holds out the promise of annual dating of recentbiospheric carbon. ¹⁴C can be measured by accelerator mass spectrometry(AMS), with results given in units of “fraction of modern carbon” (fM).The fatty ester compositions and products described herein includebioproducts that can have an fM ¹⁴C of at least about 1. For example,the bioproduct of the disclosure can have an fM ¹⁴C of at least about1.01, an fM ¹⁴C of about 1 to about 1.5, an fM ¹⁴C of about 1.04 toabout 1.18, or an fM ¹⁴C of about 1.111 to about 1.124.

Another measurement of ¹⁴C is known as the percent of modern carbon(pMC). For an archaeologist or geologist using ¹⁴C dates, AD 1950 equals“zero years old”. This also represents 100 pMC. “Bomb carbon” in theatmosphere reached almost twice the normal level in 1963 at the peak ofthermo-nuclear weapons. Its distribution within the atmosphere has beenapproximated since its appearance, showing values that are greater than100 pMC for plants and animals living since AD 1950. It has graduallydecreased over time with today's value being near 107.5 pMC. This meansthat a fresh biomass material, such as corn, would give a ¹⁴C signaturenear 107.5 pMC. Petroleum based compounds will have a pMC value of zero.Combining fossil carbon with present day carbon will result in adilution of the present day pMC content. By presuming 107.5 pMCrepresents the ¹⁴C content of present day biomass materials and 0 pMCrepresents the ¹⁴C content of petroleum based products, the measured pMCvalue for that material will reflect the proportions of the twocomponent types. For example, a material derived 100% from present daysoybeans would give a radiocarbon signature near 107.5 pMC. If thatmaterial was diluted 50% with petroleum based products, it would give aradiocarbon signature of approximately 54 pMC. A biologically basedcarbon content is derived by assigning “100%” equal to 107.5 pMC and“0%” equal to 0 pMC. For example, a sample measuring 99 pMC will give anequivalent biologically based carbon content of 93%. This value isreferred to as the mean biologically based carbon result and assumes allthe components within the analyzed material originated either frompresent day biological material or petroleum based material. Abioproduct comprising one or more fatty esters as described herein canhave a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98,99, or 100. In other instances, a fatty ester composition describedherein can have a pMC of between about 50 and about 100; about 60 andabout 100; about 70 and about 100; about 80 and about 100; about 85 andabout 100; about 87 and about 98; or about 90 and about 95. In yet otherinstances, a fatty ester composition described herein can have a pMC ofabout 90, 91, 92, 93, 94, or 94.2.

Fatty Ester Compositions

Examples of fatty esters include fatty acid esters, such as thosederived from short-chain alcohols, including FAEE and FAME, and thosederived from longer chain fatty alcohols. The fatty esters and/or fattyester compositions that are produced can be used, individually or insuitable combinations, as a biofuel (e.g., a biodiesel), an industrialchemical, or a component of, or feedstock for, a biofuel or anindustrial chemical. In some aspects, the disclosure pertains to amethod of producing a fatty ester composition comprising one or morefatty acid esters, including, for example, FAEE, FAME and/or other fattyacid ester derivatives of longer chain alcohols. In related aspects, themethod comprises a genetically engineered production host suitable formaking fatty esters and fatty ester compositions including, but notlimited to, FAME, FAEE, fatty acid propyl esters, fatty acid isopropylesters, fatty acid butyl esters, monoglycerides, fatty acid isobutylesters, fatty acid 2-butyl esters, and fatty acid tert-butyl esters, andthe like.

Esters have many commercial uses. For example, biodiesel, an alternativefuel, is comprised of esters (e.g., fatty acid methyl ester, fatty acidethyl esters, etc.). Some low molecular weight esters are volatile witha pleasant odor which makes them useful as fragrances or flavoringagents. In addition, esters are used as solvents for lacquers, paints,and varnishes. Furthermore, some naturally occurring substances, such aswaxes, fats, and oils are comprised of esters. Esters are also used assoftening agents in resins and plastics, plasticizers, flame retardants,and additives in gasoline and oil. In addition, esters can be used inthe manufacture of polymers, films, textiles, dyes, and pharmaceuticals.

In general, the fatty ester or fatty ester composition is isolated fromthe extracellular environment of the host cell. In some embodiments, thefatty ester or fatty ester composition is spontaneously secreted,partially or completely, from the host cell. In alternative embodiments,the fatty ester or fatty ester composition is transported into theextracellular environment, optionally with the aid of one or moretransport proteins. In still other embodiments, the fatty ester or fattyester composition is passively transported into the extracellularenvironment.

Fatty Alcohol Compositions

Examples of fatty alcohols include saturated-, unsaturated-,straight-chain- and branched-chain fatty alcohols. The fatty alcoholsand/or fatty alcohol compositions that are produced can be used,individually or in suitable combinations, as a detergent, an industrialchemical, or a component of, or feedstock for, an industrial chemical.In some aspects, the disclosure pertains to a method of producing afatty alcohol composition comprising one or more fatty alcohols,including, for example, shorter and longer chain fatty alcohols. Inrelated aspects, the method comprises a production host suitable formaking fatty alcohols and fatty alcohol compositions.

The methods can produce fatty alcohols comprising a C6-C26 fattyalcohol. In some embodiments, the fatty alcohol includes a C6, C7, C8,C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22,C23, C24, C25, and/or a C26 fatty alcohol. In certain embodiments, thefatty alcohol is 1-decanol, 1-dodecanol, 1-myristyl alcohol,1-hexadecanol, octadecenol, tetradecenol, or hexadecenol. In otherembodiments, the fatty alcohol includes a straight-chain fatty alcohol.In other embodiments, the fatty alcohol includes a branched-chain fattyalcohol. In yet other embodiments, the fatty alcohol comprises a cyclicmoiety. In some embodiments, the fatty alcohol is an unsaturated fattyalcohol. In other embodiments, the fatty alcohol is a monounsaturatedfatty alcohol. In yet other embodiments, the fatty alcohol is asaturated fatty alcohol. In another aspect, the invention features afatty alcohol produced by any of the methods or any of themicroorganisms described herein, or a surfactant encompassing a fattyalcohol produced by any of the methods or any of the microorganismsdescribed herein. In some embodiments, the fatty alcohol has a δ′³C ofabout −15.4 or greater. In certain embodiments, the fatty alcohol has aδ′³C of about −15.4 to about −10.9, or of about −13.92 to about −13.84.In some embodiments, the fatty alcohol has an f_(M) ¹⁴C of at leastabout 1.003. In certain embodiments, the fatty alcohol has an f_(M) ¹⁴Cof at least about 1.01 or at least about 1.5. In some embodiments, thefatty alcohol has an f_(M) ¹⁴C of about 1.111 to about 1.124.

Fatty alcohols have many commercial uses. The shorter chain fattyalcohols are used in the cosmetic and food industries as emulsifiers,emollients, and thickeners. Due to their amphiphilic nature, fattyalcohols behave as nonionic surfactants, which are useful as detergents.In addition, fatty alcohols are used in waxes, gums, resins,pharmaceutical lotions, lubricating oil additives, textile antistaticand finishing agents, plasticizers, cosmetics, industrial solvents, andsolvents for fats.

In general, the fatty alcohol or fatty alcohol composition is isolatedfrom the extracellular environment of the host cell. In someembodiments, the fatty alcohol or fatty alcohol composition isspontaneously secreted, partially or completely, from the host cell. Inalternative embodiments, the fatty alcohol or fatty alcohol compositionis transported into the extracellular environment, optionally with theaid of one or more transport proteins. In still other embodiments, thefatty alcohol or fatty alcohol composition is passively transported intothe extracellular environment.

Examples

The following specific examples are intended to illustrate thedisclosure and should not be construed as limiting the scope of theclaims.

In order to illustrate the present findings, two different methods weredeveloped to improve the native E. coli ACC enzyme for improved FAMEproduction, i.e., achieve a higher titer and yield. Although it has beenknown in the literature that increased expression of all four E. coliACC genes can improve fatty acid production, it was surprising to findthat targeted mutations in the accB gene and targeted expression changesin the accBC operon can improve FAME production.

Protocols:

1. Strain Construction for accBC

A production host strain called BD64 (supra) was used to express accBC.The production host strain contained several genetic manipulations inorder to test for expression of accBC. The chromosome region containingthe accBC operon was modified. The genetic manipulation was performed inthe presence of an ACC complementation system. Malonate was supplementedat 10 mM and simultaneously two malonate utilization genes matB and matCfrom Rhizobium trifolii were expressed from a low copy plasmid. Thesegenes were cloned behind a constitutive promoter in the pKD46integration plasmid using standard manipulation techniques. The accBCoperon was knocked out (see Datsenko et al. (2000) Proceedings of theNational Academy of Sciences 97(12):6640-6645) except that selectiveplates contained 10 mM malonate. The modified accBC operon wasintegrated using the same procedure except selective plates lackedmalonate.

2. Strain Construction for accB

A production host strain called BD64 (supra) was used to express accB.The chromosome region containing the accB gene was modified using thesame strategy used for the construction of accBC (supra).

3. ACC FAME Production Assay

Changes to the E. coli ACC enzymatic activity were assayed using a FAMEproduction system. Strain BD64 (supra) containing the desired ACCmutation(s) was transformed with an ester synthase (ES) plasmid calledpKEV13. Plasmid pKEV13 was constructed by cloning the commercial pTrcpromoter (Life Technologies) and an ester synthase gene fromMarinobacter hydrocarbonoclasticus ATCC 49840 into the plasmid pCL1920(Lerner et al. (1990) Nucleic acids research 18(15):4631.) Strains werefermented, extracted, and FAME production measured according to standardprocedures, which are detailed below.

The fermentation was performed as follows; from an LB culture growing in96 well plates 30 μL LB culture was used to inoculate 270 μL FA2P media,which was then incubated for approximately 16 hours at 32° C. in ashaking incubator. 30 μL of the overnight seed was used to inoculate 300μL FA4P media containing 2% methanol and 1 mM IPTG. Both FA2P and FA4Pmedia are modified M9 minimal media containing 0.2 g/L or 0.4 g/L(respectively) of phosphate. The carbon source in both FA2P and FA4Pmedia is 50 g/L glucose. The cultures were incubated at 32° C. in ashaking incubator for 24 hours, when they were extracted following thestandard extraction protocol detailed below.

The extraction was performed as follows; to each well to be extracted 40μL 1M HCl, then 300 μL butyl acetate with 500 mg/L C11-FAME as internalstandard was added. The 96 well plate was heat-sealed using a platesealer (ALPS-300; Abgene, ThermoScientific, Rockford, Ill.), and shakenfor 15 minutes at 2000 rpm using MixMate (Eppendorf, Hamburg, Germany).After shaking, the plate was centrifuged for 10 minutes at 4500 rpm atroom temperature (Allegra X-15R, rotor SX4750A, Beckman Coulter, Brea,Calif.) to separate the aqueous and organic layers. 50 μL of the organiclayer was transferred to a 96 well plate (96-well plate, polypropylene,Corning, Amsterdam, The Netherlands). The plate was heat sealed and thenstored at −20° C. until it was evaluated by gas chromatography flameionization detector (GC-FID).

Extraction and FAME quantification were performed as follows; 1 uL ofsample was injected onto a UFM column (cat #: UFMC00001010401, ThermoFisher Scientific, Waltham, Mass.) in a Trace GC Ultra (Thermo FisherScientific, Waltham, Mass.) with a flame ionization detector (FID). Theinstrument was set up to detect C8 to C18 FAME and quantify C12 to C18β-OH FAME.

Example 1: Mutations in accB Increase FAME Production

An error prone library of the accB gene was built and screened forvariants that showed improvement over the wild type gene. Table 3 belowdepicts a summary of the best variants. The error-prone library of theaccB gene was build using a commercially available kit (Genemorph II,Agilent Technologies). The accB gene was joined to appropriate homologyregions using the SOE PCR technique, and the library was integrated intothe E. coli chromosome as described in Protocol 1, replacing the nativeE. coli accB gene. The error-prone library was screened according toProtocol 2.

TABLE 3 Variants of accB for FAME Production Well Titer Mutations 5A02435% D2Y(TAT) K108I(ATA) 6A08 171% I10T(ACC) 2H09 155% I10T(ACC) 1G11151% I117T(ACC) P151P(CCA) 4B03 142% M44T(ACG) R84S(AGT) 2G03 135%I82N(AAC) K100N(AAC) 6E05 129% G26D(GAC) A76P(CCG) 4H09 129% R31C(GAC)I3I(ATA) 3A03 128% R31C(GAC) I3I(ATA) 5E06 127% A76V(GTG) 5G03 126%E14G(GGA) P56S(TCA) P51P(CCT) 1A09 126% I10T(ACC) 6E01 121% I78F(TTC)6F03 118% V140L(CTC) A61A(GCT) 4G09 113% E14G(GGA) P56S(TCA) P51P(CCT)5A12 111% K136I(ATA) E11E(GAA) 5F05 111% F41L (CTC) 5C07 110% A76V(GTG)5G08 109% M52V(GTG) E128D(GAC) E71E(GAG) 6H11 108% S142C(TGT) 5C10 107%A49S(TCT) T94A(GCC) M124L(TTG) T134I(ATC) A63A(GCC) A75A(GCG) P86P(CCA)

The columns of Table 3 indicate the original well location of thevariant, the FAME titer improvement over the control, and the amino acidand DNA codon changes in each variant. The results in Table 3 suggestedthat a mutation in amino acid position 2 may achieve the greatestincrease in titer. Well 5A02 showed an increase in titer of 435% ofnormal ACC activity.

Next, targeted site-saturation mutagenesis was carried out in order todetermine which individual positions and mutations provide the greatestimprovement. It was determined that indeed mutations in position 2(directly following the start codon) of accB provide the greatestincreases in FAME titer. The wild type accB contains a GAT codon atposition 2 encoding aspartic acid (Asp, D). Table 1 (supra) depicts asummary of the best variants for accB position 2. The site-saturationlibrary was build using oligonucleotide primers containing degeneratebases NNN at the second accB position. The accB gene was joined toappropriate homology regions, using the SOE PCR technique, and thelibrary was integrated into the E. coli chromosome as described inProtocol 1, replacing the native E. coli accB gene. The error-pronelibrary was screened according to Protocol 2. As can be seen in Table 1(supra), an increase in titer of up to 630% of normal ACC activity wasobserved in the mutant D2H. FIG. 5 further reflects these findings andpresents a graph that shows the FAS titer in mg/L. More specifically,the figure depicts the FAS titer (FAME) as a result of expressingvarious BCCP variants (at position 2 of the accB gene) in E. coli hostcells. WT is the control for the wild-type ACC complex. Some of theseBCCP variants improved FAS titer over 5-fold (see also Table 1). Thisfinding was surprising since BCCP variants outperformed the entire ACCcomplex in producing fatty acid derivatives. Different codons encodingthe same amino acid substitution were tested and showed that the effectwas the same, confirming that the effect of increasing malonyl-derivedcompounds, in this case fatty acid derivatives, was correlated to theamino acid change in BCCP.

Example 2: Modifying Expression of the accBC Operon Increases FAMEProduction

An expression library of the accBC operon was built and screened forvariants that showed improvement over the wild type accBC promoter.Table 2 (supra) depicts a summary of the best variants. The library wasbuilt using primers that replaced the native accBC promoter region witha bacteriophage T5 promoter library, which contained degeneratenucleotides to introduce random mutations. The T5 promoter library wasjoined to appropriate homology regions, using the SOE PCR technique, andthe library was integrated into the E. coli chromosome as described inProtocol 1, replacing the native E. coli accBC promoter. The expressionlibrary was screened according to Protocol 2. As can be seen in Table 2(supra), an increase in titer of up to 315% of normal ACC activity wasobserved with a variant promoter.

Example 3: accB and accBC Engineering can Improve the Production of anyMalonyl-CoA Derived Compound

The accB mutations (Example 1) and accBC expression changes (Example 2)can be used to increase the titer and yield of any product which isderived from malonyl-CoA. The specific mutations from Example 1 can beintroduced into any microbial strain using standard genetic manipulationtechniques. The expression of accBC can be modified in any bacterium oryeast according to Example 2 or via other methods known to those in theart, using standard genetic manipulation techniques. The operonstructure of accBC is highly conserved and found in many bacteria andother microorganisms. This will allow the same techniques to be used inseveral different organisms. Compounds derived from malonyl-CoA arenumerous and include fatty acids, fatty acid esters (FAME, FAEE, etc.),fatty alcohols, fatty amines, bifunctional fatty acids (hydroxy,diacids), bifunctional fatty alcohols, bifunctional fatty esters,bifunctional fatty amines, beta-hydroxy fatty acid derived compounds,unsaturated fatty acid-derived compounds as well as non-fatty acid basedflavanones and flavonoids, polyketides, and 3-hydroxypropionic acid.

As is apparent to one with skill in the art, various modifications andvariations of the above aspects and embodiments can be made withoutdeparting from the spirit and scope of this disclosure. Suchmodifications and variations are within the scope of this disclosure.

1.-87. (canceled)
 88. A variant operon comprising a genetically modifiedaccBC promoter that controls expression of a biotin carboxyl carrierprotein (BCCP), wherein the promoter is selected from any one of SEQ IDNOS: 93, 94, 95, and 96, wherein the operon results in an increase inBCCP expression in a recombinant microbial cell as compared to a wildtype microbial cell, and wherein the operon confers to the recombinantmicrobial cell improved production of a malonyl-CoA-derived compoundwhen compared to the corresponding wild type microbial cell.
 89. Thevariant operon of claim 88, wherein the malonyl-CoA-derived compound isa fatty acid derivative selected from the group consisting of a fattyacid, a fatty acid methyl ester (FAME), a fatty acid ethyl ester (FAEE),a fatty alcohol, a fatty amine, a beta hydroxy fatty acid derivative, abifunctional fatty acid derivative, and an unsaturated fatty acidderivative.
 90. The variant operon of claim 89, wherein themalonyl-CoA-derived compound is FAME.
 91. A recombinant microorganismcomprising the variant operon of claim
 88. 92. A method of producing amalonyl-CoA-derived compound, comprising culturing a recombinantmicroorganism in a fermentation broth containing a carbon source,wherein the recombinant microorganism comprises the variant operon ofclaim
 88. 93. A method for producing a malonyl-CoA-derived compound, themethod comprising culturing a recombinant microorganism comprising: (a)a variant biotin carboxyl carrier protein (BCCP) comprising at least onemutation in its amino acid sequence, wherein the variant BCCP comprisesa polypeptide sequence selected from the group consisting of SEQ ID NOS:4, 6, 10, 14, 16, 20, 24, 32, 48, and 70, and wherein expression of thevariant BCCP confers to a recombinant cell an increased production of amalonyl-CoA-derived compound when compared to a corresponding wild typecell; and (b) the variant operon of claim 88, in a fermentation brothcontaining a carbon source.
 94. The method of claim 92, wherein themalonyl-CoA-derived compound is a fatty acid derivative selected fromthe group consisting of a fatty acid, a fatty acid methyl ester (FAME),a fatty acid ethyl ester (FAEE), a fatty alcohol, a fatty amine, a betahydroxy fatty acid derivative, a bifunctional fatty acid derivative, andan unsaturated fatty acid derivative.
 95. The method of claim 94,wherein the malonyl-CoA-derived compound is FAME.
 96. A recombinantmicroorganism having increased expression of a nucleic acid sequencecomprising accB or accC or a combination thereof, wherein the increasedexpression is due to one or more genetically modified accBC promotersthat drive expression of the nucleic acid sequence, wherein thegenetically modified promoter is selected from any one of SEQ ID NOS:93, 94, 95, and 96, and wherein the increased expression results in anincreased production of a malonyl-CoA-derived compound when themicroorganism is cultured with a carbon source.
 97. The recombinantmicroorganism of claim 96, wherein the nucleic acid sequence encodesbiotin carboxyl carrier protein (BCCP) or biotin carboxylase (BC) or acombination thereof.
 98. The recombinant microorganism of claim 96,wherein the malonyl-CoA-derived compound is selected from the groupconsisting of a fatty acid, a fatty acid methyl ester (FAME), a fattyacid ethyl ester (FAEE), a fatty alcohol, a fatty amine, a beta hydroxyfatty acid derivative, a bifunctional fatty acid derivative, and anunsaturated fatty acid derivative.
 99. The recombinant microorganism ofclaim 98, wherein the malonyl-CoA-derived compound is FAME.
 100. Therecombinant microorganism of claim 97, wherein the microorganism isselected from the group consisting of Escherichia, Bacillus, Cyanophyta,Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus,Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces,Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus,Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas,Schizosaccharomyces, Yarrowia, and Streptomyces.
 101. The microorganismof claim 100, wherein (a) the Escherichia is Escherichia coli; (b) theCyanophyta is selected from the group consisting of Prochiorococcus,Synechococcus, Synechocystis, Cyanothece, and Nostoc punctiforme. 102.The microorganism of claim 101, wherein the Cyanophyta is selected fromthe group consisting of Synechococcus elongates PCC7942, Synechocystissp. PCC6803, and Synechococcus sp. PCC7001.
 103. A method of producing amalonyl-CoA-derived compound, comprising culturing a microorganism in afermentation broth containing a carbon source, wherein the microorganismis a microorganism of claim 96.